Application of radix aconiti carmichaeli in treatment of kidney-yang deficiency type osteoporosis

Abstract

The invention discloses application of radix aconiti carmichaeli in treatment of kidney-yang deficiency type osteoporosis, and relates to the technical field of traditional Chinese medicine. The radix aconiti carmichaeli composition for treating the kidney-yang deficiency type osteoporosis comprises a radix aconiti carmichaeli extract and a pharmaceutically acceptable carrier, the radix aconiti carmichaeli extract is prepared through a water extraction or alcohol extraction method, the content of aconite alkaloids accounts for 1%-10% of the total weight of the extract, and the composition is prepared into an oral preparation or an injection. Through the synergistic effect of dual mechanisms of anti-aging and osteogenesis promotion, the problems of single target spot and limited curative effect of the traditional medicine are effectively solved; the radix aconiti carmichaeli can improve the kidney-yang deficiency state of the mouse caused by hydrocortisone injection, meanwhile, the cAMP / cGMP ratio tends to be normal, and the pathological internal environment of kidney-yang deficiency is turned on the whole.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to the application of Aconitum carmichaelii in the treatment of osteoporosis of the kidney-yang deficiency type. Background Technology

[0002] Osteoporosis (OP) is a systemic degenerative bone disease characterized by decreased bone mass, destruction of bone microstructure, increased bone fragility, and increased risk of fractures. A 2018 epidemiological survey of osteoporosis in my country showed that the prevalence of osteoporosis in people over 65 years of age reached 32.0%, with a prevalence of 10.7% in men and 51.6% in women. The fracture rate in osteoporosis increases with age, reaching as high as 37% in people over 80 years of age. Currently, Western medicine treatments for osteoporosis mainly rely on hormone drugs and hormone replacement therapy, which, while effective, have adverse reactions and are expensive. Traditional Chinese medicine, as a treatment for osteoporosis, has the unique advantage of multi-component, multi-target synergistic therapy.

[0003] In recent years, Traditional Chinese Medicine (TCM) has demonstrated unique advantages in the prevention and treatment of osteoporosis, attracting increasing attention from researchers. Aconite, as one of the traditional Chinese medicines used to warm the Yang, has the effects of restoring Yang and rescuing from collapse, and is often used to treat diseases related to Yang deficiency and cold excess. Modern pharmacological studies have found that aconite contains various active ingredients, such as aconitine alkaloids, polysaccharides, and volatile oils, possessing multiple biological activities including anti-inflammatory, antioxidant, immunomodulatory, and bone metabolism-promoting effects. Especially in osteoporosis research, aconite has shown good anti-osteoporosis potential, and its mechanism of action may involve promoting osteoblast differentiation and bone formation, inhibiting osteoclast activity, and improving the bone marrow microenvironment. TCM typically considers the pathogenesis of osteoporosis (OP) based on its main symptoms and clinical manifestations to be primarily due to spleen and kidney deficiency, resulting in insufficient bone marrow biochemistry and malnourishment of the bones, leading to weakness and atrophy. Spleen and kidney Yang deficiency is the most common, with kidney Yang deficiency type osteoporosis accounting for a large proportion. Clinically, kidney-tonifying Yang herbs are commonly used for prevention and treatment. In common classic formulas for tonifying kidney yang, aconite is used as the chief herb, sometimes supplemented with dried ginger and licorice for detoxification, or with yin-nourishing herbs, to treat kidney yang deficiency OP. "Aconite is not warming without ginger," meaning that dried ginger synergistically enhances the effects of aconite while effectively reducing its toxicity.

[0004] Furthermore, aging is considered a significant risk factor for the development and progression of osteoporosis. With increasing age, the body's oxidative stress levels rise, cellular senescence accelerates, and the ability of bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteogenic cells weakens, leading to a decline in bone formation capacity. Studies have shown that Aconitum carmichaelii (Fuzi) can improve the condition of the elderly by scavenging free radicals, enhancing the activity of antioxidant enzymes, and delaying cellular senescence, thereby improving osteogenic function. Some animal and cell experiments have also confirmed that Aconitum carmichaelii or its extracts can significantly increase bone mineral density and improve trabecular bone structure in aging-related models, suggesting that it possesses an anti-aging mechanism against osteoporosis.

[0005] In conclusion, Aconitum carmichaelii, a traditional Chinese medicine with warming and tonifying kidney yang, anti-aging, and bone metabolism-promoting effects, shows promising application prospects in the prevention and treatment of osteoporosis. Further research into the specific mechanisms by which Aconitum carmichaelii treats osteoporosis through its anti-aging and osteogenic effects is expected to provide a scientific basis for its clinical application and offer new theoretical support and treatment strategies for traditional Chinese medicine intervention in osteoporosis. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of Aconitum carmichaelii in the treatment of osteoporosis of the kidney-yang deficiency type.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The application of Aconitum carmichaelii in the treatment of osteoporosis of kidney-yang deficiency type is characterized by the extraction of Aconitum carmichaelii extract and its application in the preparation of drugs for the treatment of osteoporosis of kidney-yang deficiency type.

[0008] An aconite composition for treating osteoporosis of the kidney-yang deficiency type, comprising an aconite extract and a pharmaceutically acceptable carrier, wherein the aconite extract is prepared by water extraction or alcohol extraction, wherein the aconite alkaloid content accounts for 1%-10% of the total weight of the extract, and the composition is formulated as an oral preparation or injection.

[0009] Preferably, the aconite composition further comprises a dried ginger extract, wherein the weight ratio of the aconite extract to the dried ginger extract is 1:0.5 to 1:2, wherein the dried ginger extract is used to reduce the toxicity of aconite and enhance the osteogenic effect through synergistic action.

[0010] Preferably, the dried ginger extract is obtained by ethanol reflux extraction, wherein the gingerol content is not less than 3%, and the ratio is 1:1.

[0011] Preferably, it further comprises an osteogenic accelerator, said osteogenic accelerator being one or more of calcium, vitamin D, or sodium alendronate, wherein the weight ratio of Aconitum carmichaelii extract to osteogenic accelerator is 1:0.1 to 1:5.

[0012] A method for preparing the above-mentioned Aconitum carmichaelii composition includes the following steps: after the Aconitum carmichaelii raw material is pulverized, it is extracted by water extraction, the amount of water added is 6-10 times the weight of the medicinal material, and the extracts are combined and concentrated to a clear extract with a relative density of 1.10-1.15.

[0013] Preferably, the water extraction method further includes an alcohol precipitation step: ethanol is added to the concentrated extract to make the alcohol content reach 60%-70%, and after standing for 24 hours, the supernatant is taken, and the ethanol is recovered to obtain the Aconitum carmichaelii extract.

[0014] Preferably, the water extraction method uses an extraction temperature of 80-100℃, an extraction time of 1-2 hours, and is performed 2-3 times.

[0015] A quality testing method for the aconite composition as described above includes the following test indicators: the content of aconite alkaloids is determined by HPLC, and the total content of aconitine, neoaconitine, and hypoaconitine is not less than 1.5%; the microbial limit test meets the pharmacopoeia requirements; and the heavy metal content does not exceed 10 ppm.

[0016] A pharmaceutical package comprising the Aconitum carmichaelii composition as described above, and accompanied by an instruction manual describing the composition, efficacy, usage, dosage, and precautions of the drug.

[0017] A diagnostic and drug selection system for osteoporosis of kidney-yang deficiency type includes a TCM syndrome scoring module and a drug ratio recommendation module, which automatically recommends the optimal compatibility ratio and dosage of the above-mentioned Aconitum carmichaelii combination based on the patient's syndrome score.

[0018] The beneficial effects of this invention are as follows: 1. This invention effectively solves the problem of single target and limited efficacy of traditional drugs through the synergistic effect of anti-aging and bone-promoting dual mechanisms; Aconitum carmichaelii can improve the kidney yang deficiency state in mice induced by hydrocortisone injection, and at the same time, the cAMP / cGMP ratio tends to normalize, thus reversing the pathological internal environment of kidney yang deficiency as a whole.

[0019] 2. This invention clarifies the compatibility ratio of Aconitum carmichaelii and dried ginger, which not only utilizes the synergistic effect of Aconitum carmichaelii being less heating without ginger, but also effectively reduces the toxicity of Aconitum carmichaelii through dried ginger, ensuring the safety of clinical application; making the drug efficacy stable and highly reproducible.

[0020] 3. The composition of the present invention can significantly improve bone microstructure. After treatment, bone mineral density, bone volume fraction, number and thickness of trabeculae are significantly improved, while trabecular separation is reduced, which is visually demonstrated by imaging to improve bone quality. Attached Figure Description

[0021] Figure 1 The figure shows the effect of Aconitum carmichaelii on the body weight of OP mice with kidney yang deficiency in this invention; Figure 2 This is a diagram illustrating the effect of Aconitum carmichaelii on kidney yang deficiency in OP mice according to the present invention. Figure 3 This is a diagram showing the effect of Aconitum carmichaelii on bone mass and bone microstructure in RA-induced osteoporotic mice in this invention. Figure 4 This is a schematic diagram illustrating how Aconitum carmichaelii alleviates femoral aging and promotes osteogenic formation in RA-induced osteoporosis in mice, as described in this invention. Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0023] Example 1: 1 Materials and Methods 1.1 Experimental Materials 1.1.1 Animals Forty 8-week-old female C57BL / 6J mice (SPF grade, SCXK, 2022-0010) were provided by the Experimental Animal Center of Chongqing Medical University. This animal research protocol was reviewed and approved by the Medical Ethics Committee of Chongqing Medical University (Approval No.: IACUC-CQMU-2024-11024).

[0024] 1.1.2 Main Drugs and Reagents Aconitum carmichaelii and alendronate sodium, used as positive controls, demonstrated clear therapeutic effects on osteoporosis (OP) in various animal models, increasing bone mineral density and improving trabecular microstructure. Other treatments included retinoic acid (RA), hydrocortisone, antibodies (P16, P21, ALP, OCN), anhydrous ethanol, and hematoxylin-eosin staining.

[0025] 1.1.3 Main Instruments Pathology slide cutter, ELISA reader, upright microscope, electrophoresis apparatus, electrophoresis tank, etc.

[0026] 1.2 Experimental Methods 1.2.1 Animal grouping and model establishment: C57BL / 6J mice were randomly divided into 4 groups: blank group, retinoic acid group (model group), positive group, and Aconitum carmichaelii group. Except for the blank group, all other groups of mice were administered retinoic acid by gavage (90 mg / kg). -1 A mouse model of osteoporosis was established after two weeks of treatment. Simultaneously, a kidney-yang deficiency type of osteoporosis model was established by intramuscular injection of hydrocortisone (25 mg / kg) for 10 consecutive days. After model establishment, medication was initiated in the third week. The control group and model group were administered physiological saline by gavage, while the remaining groups were administered 0.2 mL / day of the corresponding concentration of traditional Chinese medicine solution by gavage. The mice were weighed before each day's administration to adjust the gavage volume. Hydrocortisone was injected twice weekly to maintain the kidney-yang deficiency state until the end of the treatment. These animals were fed under standard conditions and subjected to a 12-hour regulated light / dark cycle.

[0027] 1.2.2 Sample collection: The mice were fasted for 12 hours before blood collection but allowed to drink water. After blood collection, the mice were allowed to stand at room temperature and centrifuged at 3000 rpm for 10 minutes. The upper serum layer was collected and stored at -80℃ for later use. The femurs of both sides of the mice were removed and fixed in paraformaldehyde.

[0028] 1.3 Detection Indicators and Methods 1.3.1 Detection indicators and methods for kidney yang deficiency: The syndrome of mice was evaluated according to the basic theory of traditional Chinese medicine, and the kidney yang deficiency syndrome of mice was scored according to the "Experimental Methodology of Syndrome Differentiation and Treatment"; cAMP and cGMP content were detected by ELISA.

[0029] Kidney Yang Deficiency Syndrome Score: 1.3.2 Osteoporosis detection indicators and methods The left femur of the mouse was first decalcified, sectioned, and stained with hematoxylin and eosin (HE). The specific operation was as follows: paraffin sections were dewaxed to water: in environmentally friendly dewaxing agent (1), environmentally friendly dewaxing agent (2), and environmentally friendly dewaxing agent (3) for 10 minutes each, then in anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes each. Rinse with tap water for 1 minute; stain with hematoxylin staining solution (Harris) for 4 minutes, rinse with tap water for 2 minutes until no excess staining solution was removed from the section; differentiate with 0.8% hydrochloric acid alcohol for 2 seconds, rinse with tap water, or use lithium carbonate aqueous solution to return to blue, then rinse with water for 2 minutes; stain with eosin staining solution (alcohol-soluble) for 20 seconds, no water rinsing is required, directly immerse in 95% ethanol for 5 seconds to adjust the color, immerse in anhydrous ethanol (1) and anhydrous ethanol (2) for 2 minutes to dehydrate; finally, clear with environmentally friendly transparent agent, seal, and examine under a microscope.

[0030] Micro-CT scans were performed on the right femur of the mouse. The specific procedure was as follows: (1) μCT Scan: Micro-CT scans were performed on all sampled specimens after fixation. The scanning parameters were: X-ray tube current 200uA, voltage 60KV, scanning the entire object, scanning resolution 6.554883μm, exposure time 205ms, and scanning angle 180 degrees. A phantom (provided by the equipment manufacturer with standardized configuration) was calibrated under the same conditions. The original images were obtained after scanning.

[0031] (2) μCT Reconstruction: The original image was reconstructed using the 3D reconstruction software NRecon (software version V1.7.4.2, Bruker GmbH, Germany). Before reconstruction, the reconstructed image was previewed. To reduce image artifacts and achieve the best reconstruction effect, the reconstruction parameters were adjusted and set to the following values: Smoothing=5, Beam-hardening=8, Ringartifacts=25%, etc. After setting the parameters, the specified folder was selected and image reconstruction was started.

[0032] (3) μCT analysis: The region of interest (ROI) was analyzed using CTAnalyser (software version 1.20.3.0, Bruker GmbH, Germany). Uniform parameters were set, and the software calculated parameters such as total tissue volume (TV), bone volume (BV), volume ratio (BV / TV), bone area (BS), trabecular bone number (Tb.N), and trabecular bone thickness (Tb.th).

[0033] 1.3.3 Enzyme-linked immunosorbent assay (ELISA) Serum stored at -80℃ was used, and its concentration was detected using an ELISA kit containing type I procollagen n-terminal propeptide (P1NP, Elabscience, E-EL-M0233, Wuhan, China) and type I collagen C-terminal propeptide (β-CTX, Elabscience, NE-EL-R1405, Wuhan, China). The specific procedure was as follows: (1) Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips in a self-sealing bag and put them back at 4C.

[0034] (2) Set up standard wells and sample wells, and add 50 pL of standard at different concentrations to each standard well.

[0035] (3) Add 10 μL of the sample to be tested to the sample well first, and then add 40 μL of sample diluent; do not add to the blank well.

[0036] (4) Except for the blank wells, add 100uL of horseradish peroxidase (HRP) labeled detection antibody to each of the standard wells and sample wells, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 60min.

[0037] (5) Discard the liquid, pat dry on absorbent paper, fill each well with washing liquid, let stand for 1 minute, shake off the washing liquid, pat dry on absorbent paper, and repeat the washing process 5 times (or use a plate washer).

[0038] (6) Add 50 L of substrate A and 50 L of substrate B to each well and incubate at 37°C in the dark for 15 min. (7) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0039] 1.3.4 Western Blot analysis of ALP and OCN protein expression in the femur The femurs of each group of mice were ground in a mortar with liquid nitrogen, followed by the addition of protease phosphatase inhibitor and RPA lysis buffer. Protein concentration was measured using the BCA method, and the protein was denatured at 100°C for 15 min. After protein loading, electrophoresis, electroporation, and blocking with 5% skim milk powder at room temperature for 1 h, the membrane was incubated overnight at 4°C with GAPDH (1:20000), ALP (1:2000), OCN (1:2000), P16 (1:2000), and P21 (1:2000) antibodies. After washing with TBST, the membrane was incubated with secondary antibody (1:10000) at room temperature for one hour, followed by washing again. Then, ECL exposure and development were performed, and the grayscale value was detected using ImageJ.

[0040] 1.4 Statistical Analysis One-way ANOVA was used to compare differences between groups. If homogeneity of variance was established, Bonferroni correction was applied for statistical analysis; if variances were unequal, Tamhane's t² test was used. A p-value < 0.05 was considered statistically significant. Furthermore, statistical charts were generated using GraphPad Prism 10.1.2 software.

[0041] 2 Results I. The combination of dried ginger and aconite can rescue the weight loss in a mouse osteoporosis model induced by retinoic acid.

[0042] like Figure 1 The following chart shows the effect of Aconitum carmichaelii on the body weight of OP mice with kidney-yang deficiency. A. Overall body weight change of mice. B. Mouse weight on the last day after modeling. C. Mouse weight on day 5 after drug administration. D. Mouse weight on day 10 after drug administration. E. Mouse weight after two weeks of drug administration. Statistical significance is expressed as: compared with the control group. ### p < 0.001; compared with the RA group * p<0.05.

[0043] II. Aconite can improve the kidney yang deficiency state in mice induced by hydrocortisone injection.

[0044] like Figure 2The following shows the effect of Aconitum carmichaelii on kidney-yang deficiency in OP mice. A. Serum cAMP (cyclic adenosine monophosphate) content in kidney-yang deficiency OP mice; B. Serum cGMP (cyclic guanosine monophosphate) content in kidney-yang deficiency OP mice; C. cAMP / cGMP ratio. Statistical significance is expressed as: compared with the control group. ### p < 0.001 # p < 0.05; compared with the RA group *** p < 0.001 * p<0.05.

[0045] III. Aconitum carmichaelii can restore bone microstructural abnormalities in RA-induced osteoporosis mice.

[0046] like Figure 3 As shown, the effects of Aconitum carmichaelii on bone mass and bone microstructure in RA-induced osteoporosis mice are: A. Serum calcium content in OP mice with kidney-yang deficiency. B. Representative H&E stained images of the femur in each group (arrows indicate the morphology of trabeculae) (scale bar: 500µm). C. Representative micro-CT images of distal femur trabeculae in each group. Quantitative analysis of trabecular parameters showed that Aconitum carmichaelii treatment improved (D) bone mineral density (BMD), (E) bone volume fraction (BV / TV), (F) number of trabeculae (Tb.N), (G) trabecular thickness (Tb.Th), and (H) trabecular separation (Tb.Sp). Statistical significance is expressed as: compared with the control group. ### p < 0.001 ## p < 0.01; compared with the RA group ** p < 0.01 * p<0.05.

[0047] IV. Aconite can slow down femoral aging and promote osteogenic formation in OP mice with kidney yang deficiency.

[0048] like Figure 4 As shown, Aconitum carmichaelii can alleviate femoral aging and promote osteogenic formation in RA-induced osteoporosis in mice. A, C, and D show increased levels of osteogenic markers in the femur of OP mice treated with Aconitum carmichaelii; B, E, and F show decreased protein levels of aging markers P16 and P21 through Western blot and optical density analysis. Statistical significance is expressed as: compared with the control group. ### p < 0.001 ## p < 0.01; compared with the RA group ** p < 0.01 * p<0.05.

[0049] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of Aconitum carmichaelii in the treatment of osteoporosis of kidney-yang deficiency type, characterized in that, Aconite extract was extracted and applied to the preparation of drugs for the treatment of osteoporosis of the kidney-yang deficiency type.

2. An Aconitum carmichaelii composition for treating osteoporosis of the kidney-yang deficiency type, characterized in that, The composition comprises an aconite extract and a pharmaceutically acceptable carrier, the aconite extract being prepared by water extraction or alcohol extraction, wherein the aconite alkaloid content accounts for 1%-10% of the total weight of the extract, and the composition is formulated as an oral or injectable preparation.

3. The Aconitum carmichaelii composition according to claim 2, characterized in that, The aconite composition further comprises a dried ginger extract, wherein the weight ratio of the aconite extract to the dried ginger extract is 1:0.5 to 1:2, wherein the dried ginger extract is used to reduce the toxicity of aconite and enhance the osteogenic effect through synergistic action; the dried ginger extract is obtained by ethanol reflux extraction, wherein the gingerol content is not less than 3%, and the compatibility ratio is 1:

1.

4. The Aconitum carmichaelii composition according to claim 3, characterized in that, It also contains an osteogenic accelerator, which is one or more of calcium, vitamin D, or sodium alendronate, wherein the weight ratio of Aconitum carmichaelii extract to osteogenic accelerator is 1:0.1 to 1:

5.

5. A method for preparing an Aconitum carmichaelii composition according to any one of claims 1-4, characterized in that, Includes the following steps: After being crushed, the raw aconite is extracted using a water extraction method. The amount of water added is 6-10 times the weight of the medicinal material. The extracts are then combined and concentrated to a clear paste with a relative density of 1.10-1.

15.

6. The preparation method according to claim 5, characterized in that, The water extraction method also includes an alcohol precipitation step: ethanol is added to the concentrated extract to make the alcohol content reach 60%-70%, and after standing for 24 hours, the supernatant is taken, and the ethanol is recovered to obtain the Aconitum carmichaelii extract.

7. The application of Aconitum carmichaelii in the treatment of osteoporosis of kidney-yang deficiency type according to claim 5, characterized in that, The water extraction method involves an extraction temperature of 80-100℃, an extraction time of 1-2 hours, and 2-3 extractions.

8. A method for quality testing of the Aconitum carmichaelii composition as described in any one of claims 1-4, characterized in that, The following testing indicators are included: the content of aconitine alkaloids is determined by HPLC, and the total content of aconitine, neoaconitine, and hypoaconitine is not less than 1.5%; the microbial limit test complies with the pharmacopoeia; and the heavy metal content does not exceed 10 ppm.

9. A drug packaging, characterized in that, The product comprises the Aconitum carmichaelii composition as described in any one of claims 1-4, and is accompanied by an instruction manual describing the composition, efficacy, usage, dosage, and precautions of the drug.

10. A diagnostic and drug selection system for osteoporosis of kidney-yang deficiency type, characterized in that, It includes a TCM syndrome scoring module and a drug ratio recommendation module, which automatically recommends the optimal compatibility ratio and dosage of the Aconitum carmichaelii composition according to any one of claims 1-4 based on the patient's syndrome score.

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