Mongolian medicine compound for treating fragile fracture and application thereof

Abstract

The invention discloses a Mongolian medicine compound for treating fragile fracture and application of the Mongolian medicine compound, and belongs to the field of biological medicine. The Mongolian medicine compound comprises the following raw material components in parts by weight: 5-20 parts of pomegranate, 5-10 parts of tribulus terrestris, 10-15 parts of fructus piperis longi, 2-20 parts of fructus alpiniae oxyphyllae, 5-15 parts of cluster mallow fruit, 5-15 parts of cinnamon, 5-15 parts of rhizoma polygonati, 5-20 parts of safflower, 5-15 parts of radix asparagi, 5-15 parts of radix polygonati officinalis and 5-20 parts of radix trichosanthis. Clinical verification proves that the Mongolian medicine compound has a good treatment effect on the fragile fracture, animal experiments prove that the medicine composition can improve the bone microstructure of rats and adjust bone metabolism, the effect of treating the fragile fracture is further achieved, and a theoretical support is provided for developing the Mongolian medicine for treating the fragile fracture.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a Mongolian medicine compound for treating brittle fractures and application thereof. Background Art

[0002] Fragility fractures are serious systemic bone diseases characterized by decreased bone mass and deterioration of bone tissue structure, which increase bone brittleness and eventually form fractures. The most common brittle fractures occur in the hip, wrist and lumbar spine. Bone mineral density (BMD) is a key tool for identifying osteoporosis. Its introduction into clinical practice 30 years ago was an important step forward in the development of the medical industry. The WHO reported that a BMD T value ≤-2.5 is defined as osteoporosis, which in turn leads to brittle fractures. Fragile fractures are mainly caused by insufficient hormone secretion. In recent years, significant progress has been made in the study of factors related to the occurrence of fragility fractures. Studies have shown that age, body mass index, diet, exercise and genetics are closely related to the occurrence of fragility fractures.

[0003] At present, the treatment of brittle fractures is mainly through drug therapy with Western medicine compounds. Although this kind of therapeutic drug has a certain therapeutic effect, it also has many toxic side effects and is prone to cause other complications. In addition, the medical cost of Western medicine treatment is high, expensive, and patient compliance is poor. Mongolian medicine has local characteristics in the treatment of bone metabolic diseases, and has few toxic side effects, which is in line with the "green medical" concept pursued by people. Therefore, following the theory of Mongolian medicine and developing drugs that can be used to treat brittle fractures is of great significance to enrich the medical strategy of the disease. Summary of the invention

[0004] The purpose of the present invention is to provide a Mongolian medicine compound for treating brittle fractures and its application, so as to solve the problems existing in the above-mentioned prior art. The Mongolian medicine compound of the present invention has a wide range of raw materials, low cost, and small drug toxicity and side effects, has a significant effect on treating brittle fractures, and provides theoretical support for the development of Mongolian medicine for treating brittle fractures.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a pharmaceutical composition for treating brittle fractures. The raw materials include the following components in parts by weight: 5-20 parts of pomegranate, 5-10 parts of tribulus terrestris, 10-15 parts of piper longum, 5-20 parts of alpinia oxyphylla, 5-15 parts of malvaceae fruit, 5-15 parts of cinnamon bark, 5-15 parts of polygonatum, 5g-20 parts of safflower, 5-15 parts of asparagus, 5-15 parts of polygonatum odoratum and 5-20 parts of trichosanthes.

[0007] Optionally, the raw materials, measured by weight, include the following components: 10 parts of pomegranate, 5 parts of tribulus terrestris, 15 parts of piper longum, 20 parts of alpinia oxyphylla, 5 parts of okra fruit, 10 parts of cinnamon bark, 10 parts of polygonatum, 20 parts of safflower, 5 parts of asparagus, 10 parts of polygonatum, and 5 parts of trichosanthes.

[0008] The present invention also provides a method for preparing the pharmaceutical composition, comprising the steps of mixing the raw materials by weight, boiling with water and concentrating to obtain the pharmaceutical composition.

[0009] Optionally, the amount of water added is 10 times the weight of the raw materials after mixing.

[0010] Optionally, the decocting time is 45 minutes.

[0011] Optionally, the decoction is concentrated to 15% to 35% of the original volume.

[0012] The present invention also provides application of the pharmaceutical composition in preparing a medicine for treating brittle fractures.

[0013] The present invention also provides application of the pharmaceutical composition in preparing a drug for improving osteoporosis.

[0014] The present invention also provides application of the pharmaceutical composition in preparing a drug for regulating bone metabolic balance.

[0015] Optionally, the dosage form of the drug includes pills, powders, tablets and granules.

[0016] The present invention discloses the following technical effects:

[0017] The present invention is guided by the Mongolian medicine theory of "clear and turbid metabolism", and selects pomegranate, tribulus terrestris, piper longum, alpinia oxyphylla, malva fruit, cinnamon bark, polygonatum sibiricum, safflower, asparagus cochinchinensis, polygonatum odoratum and trichosanthes as raw materials to prepare a pharmaceutical composition. Clinical verification has confirmed the good therapeutic effect of the pharmaceutical composition on brittle fractures, and further animal experiments have confirmed that the pharmaceutical composition of the present invention can improve the bone microstructure of rats, regulate bone metabolism, and thus play a role in treating brittle fractures.

[0018] The pharmaceutical composition of the present invention has a wide range of raw material sources, low cost, and little drug toxicity and side effects, has a significant therapeutic effect on the treatment of brittle fractures, and provides a theoretical support for the development of Mongolian medicine for the treatment of brittle fractures. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 Micro-CT scanning images of the distal femur of rats in each group;

[0021] Figure 2 Results of topological data related to bone microstructure of rats in each group, including Tb.Sp (A), BV / TV (B), BMD (C), Tb.Th (D), DA (E) and Tb.N (F);

[0022] Figure 3 The biomechanical test results of the bones of rats in each group; A is the maximum stiffness, B is the maximum displacement, and C is the maximum load;

[0023] Figure 4 The femoral tissue pathological staining results of rats in each group (100×);

[0024] Figure 5 The ELISA test results of serum bone metabolism indexes ALP (A) and TRAP (B) of rats in each group;

[0025] Figure 6 The WB test results of osteogenic-related proteins in rats of each group;

[0026] Figure 7 The immunohistochemical detection of Runx2 protein in femoral tissue of rats in each group (A) and the statistical results of expression levels (B);

[0027] Figure 8 The immunohistochemical detection images of OCN protein in femoral tissue of rats in each group (A) and the statistical results of expression levels (B);

[0028] Fig. 9 Fluorescence images of the changes in mitochondrial membrane potential detected in rats of each group;

[0029] Fig.10 NAD is the liver metabolism index of rats in each group + / NADH(A) and Na + -K + -ATPase activity (B) t-statistic graph. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] According to the Mongolian medicine theory of "clear and turbid metabolism", postmenopausal women and the elderly have an imbalance of Yihei, Xila and Badagan (collectively referred to as the three roots), and the characteristics of the three roots become abnormal, causing Badaganhei to accumulate in the body, weakening the spleen and stomach, reducing stomach fire, and causing clear and turbid metabolism dysfunction, leading to clear and turbid metabolism disorders of "food essence, blood, muscle, fat, bone, bone marrow, and vitality element (collectively referred to as the seven elements)", "the separation of essence and dregs is blocked or the essence is mixed with turbid matter", especially "muscle fat essence cannot nourish bone and bone marrow essence", resulting in bone nutrition deficiency, bone reduction, decreased bone strength, and bone essence mixed with turbid matter, leading to bone clear and turbid metabolism disorders, and ultimately the formation of an increase in bone Badaganhei and brittle fractures.

[0036] Guided by the above theory, pomegranate, tribulus terrestris, piper longum, alpinia oxyphylla, winter mallow fruit, cinnamon bark, polygonatum sibiricum, safflower, asparagus cochinchinensis, polygonatum odoratum and root of trichosanthes are selected as raw materials to prepare a pharmaceutical composition. In parts by weight, the amount of each raw material is 5g-20g of pomegranate, 5g-10g of tribulus terrestris, 10g-15g of piper longum, 5g-20g of alpinia oxyphylla, 5g-15g of winter mallow fruit, 5g-15g of cinnamon bark, 5g-15g of polygonatum sibiricum, 5g-20g of safflower, 5g-15g of asparagus cochinchinensis, 5g-15g of polygonatum odoratum and 5g-20g of root of trichosanthes.

[0037] According to the composition and content of the above-mentioned pharmaceutical composition, various raw materials are mixed, ten times the weight of water is added and boiled for 45 minutes, and then concentrated to 15% to 35% of the original volume by a rotary evaporator to prepare a pharmaceutical composition.

[0038] Optionally, the above pharmaceutical composition can be processed into different dosage forms such as pills, powders, tablets, granules, etc.

[0039] Clinical verification and animal experiment verification have shown that the pharmaceutical composition prepared according to the above composition and content has shown good therapeutic effect on brittle fractures.

[0040] In a specific embodiment, the amount of the above raw materials is 10 parts of pomegranate, 5 parts of tribulus terrestris, 15 parts of piper longum, 20 parts of alpinia oxyphylla, 5 parts of wintergreen fruit, 10 parts of cinnamon, 10 parts of polygonatum, 20 parts of safflower, 5 parts of asparagus, 10 parts of polygonatum, and 5 parts of trichosanthes.

[0041] Unless otherwise specified, the reagents and materials used in the embodiments of the present invention can be obtained through conventional purchase, and the detection methods and calculation methods involved are conventional methods in the art.

[0042] The following specific examples demonstrate the therapeutic effect of the pharmaceutical composition of the present invention on brittle fractures.

[0043] Example 1 Clinical validation of the pharmaceutical composition for treating fragility fractures

[0044] This example uses a randomized controlled study to verify the clinical efficacy of the drug composition by observing changes in the pain visual analogue scale (VAS), functional disability index (ODI), Mongolian medicine symptom score, typical symptoms, quality of life, bone density (BMD), bone metabolism markers, serum indicators, liver and kidney function, etc., and evaluates the purpose of treating brittle fractures by regulating the "clear and turbid metabolism" function and inhibiting bone Bada Ganheyi.

[0045] 1. Sample estimation and case sources

[0046] The Power and Sample Size software was used to estimate the sample size. The optimal sample size was 38 people per group. The cases were randomly divided into an observation group and a control group. The two groups were enrolled in a 1:1 ratio. Considering the dropout rate of 5-10%, 45 people were set in each group. From January 1, 2022 to December 30, 2022, 90 patients who were diagnosed with PMOP in the Mongolian Medicine and Traumatology Department and Internal Medicine Clinic of the Mongolian Medicine and Traditional Chinese Medicine Hospital Affiliated to Inner Mongolia Medical University were selected and signed informed consent. Approved by the Medical Ethics Committee of the Mongolian Medicine and Traditional Chinese Medicine Hospital Affiliated to Inner Mongolia Medical University.

[0047] 1. Diagnostic criteria for case selection: The diagnostic criteria for fragility fractures are in line with the diagnostic criteria described in the 2019 edition of the "Guidelines for the Diagnosis and Treatment of Bone Atrophy with Traditional Chinese Medicine": DXA is used to measure the bone density values ​​of the lumbar spine, femoral neck and total hip in three areas. A bone density T value that is 1 standard deviation or less lower than the peak bone mass of healthy young people of the same gender is normal, a value lower than 1.0 to 2.5 standard deviations is low bone mass (or low bone mass), and a value lower than 2.5 standard deviations is accompanied by one or more brittle fractures.

[0048] 2. The included patients must meet the diagnostic criteria for bone Bada Ganheyi fragility fractures in the Mongolian Medical Diagnosis and Treatment Methods for 18 Specialties and 49 Diseases edited by Burenbatu. The main symptoms of Mongolian medicine: back pain, distal radius fracture or lumbar fracture or hip fracture; the secondary symptoms of Mongolian medicine: poor appetite, insomnia, dreaminess, diarrhea, sallow complexion, pale tongue, white fur, weak pulse, etc. The diagnosis can be confirmed if the main symptoms are present and two or more secondary symptoms are present.

[0049] 3. Inclusion criteria for case selection: meeting the diagnostic criteria for (bone atrophy) and bone fragility fractures; postmenopausal patients aged 55 to 68 years, with natural menopause >5 years; accompanied by distal radius fracture, lumbar fracture or hip fracture; patients and their families signed informed consent and voluntarily participated.

[0050] 4. Exclusion criteria for case selection: patients with non-natural menopause or pre-menopause after ovarian or hysterectomy; patients with secondary or idiopathic osteoporosis, bone tuberculosis, or bone tumors; patients with liver and kidney dysfunction, abnormal blood calcium, phosphorus, alkaline phosphatase, or mental illness; patients who have received anti-osteoporosis treatment within six months before treatment.

[0051] 5. Dropout and discontinuation criteria: poor compliance of the subjects, failure to take medication as prescribed; loss of follow-up; incomplete clinical data, affecting the results.

[0052] 6. Criteria for termination of the trial: The subject experiences serious adverse reactions; the patient refuses further treatment and withdraws from the trial on his / her own initiative.

[0053] 2. Research Methods

[0054] 1. Case screening and grouping

[0055] A total of 90 cases were selected according to the inclusion and exclusion criteria. The general relevant information such as the name of the subject, medical record number, group, start time, and completion time were collected. The 90 patients were randomly divided into observation group and control group using the random number table method: 45 cases in each group.

[0056] 2. Treatment plan

[0057] Drugs: calcium carbonate D3 tablets (600 mg / tablet), alendronate sodium tablets (10 mg / tablet) and the pharmaceutical composition prepared by the present invention (pills, 0.2 g / pill).

[0058] Basic treatment: Give calcium carbonate D3 tablets, 2 hours after meals, once a day, 1 tablet each time, with warm water.

[0059] (1) Control group treatment: basic treatment + alendronate sodium tablets (oral, take on an empty stomach 30 minutes before breakfast every day, with warm water, once a day, 1 tablet each time).

[0060] (2) Treatment of the observation group: basic treatment + drug combination pills (oral, 1 hour after meals, with warm water or brown sugar water, 3 g / time, 3 times a day).

[0061] One course of treatment is 3 months, and the medication should be taken continuously for 2 courses.

[0062] The pharmaceutical composition pills are obtained by drying the pharmaceutical composition of the present invention and then crushing it. The powder and water are stirred to form small pellets with a diameter of about 0.5-1 mm. The pellets are placed in a coating pan while adding medicine and spraying water. When the pellets finally have a diameter of about 0.5 mm, screening is started to remove oversized, undersized and irregular-shaped pellets, and then drying is performed to obtain the pellets.

[0063] 3. Observation indicators

[0064] General indicators: collect indicators such as subject age, body mass index, menopause time, waist circumference, etc.

[0065] Key indicators:

[0066] The visual analogue scale (VAS) score of pain was evaluated on the day before treatment, the second day after one course of treatment, and the second day after two courses of treatment.

[0067] The Oswestry disability index (ODI) score was evaluated on the day before treatment, the second day after one course of treatment, and the second day after two courses of treatment.

[0068] The Mongolian medicine symptom score was quantitatively scored with reference to the Mongolian medicine main symptoms of Bada Ganheyi fragility fracture in "Mongolian Medicine Diagnosis and Treatment Methods for 18 Specialties and 49 Diseases" edited by Buren Batu: low back pain, distal radius fracture or lumbar fracture or hip fracture, and secondary symptoms: poor appetite, insomnia, etc. The patients were evaluated on the day before treatment and on the second day after 1 and 2 courses of treatment.

[0069] Typical symptoms: spleen and stomach discomfort, soreness and weakness in the waist and knees, difficulty walking, lower limb cramps, difficulty holding weight, etc. were quantitatively scored. The patients were quantitatively scored on the day before treatment and the second day after 1 and 2 courses of treatment.

[0070] Quality of life: Quantitative scores were given for mental health (MH), social function (SF), role emotion (RE), energy (VT), general health (GH), bodily pain (BP), role physiology (RP), and physical function (PF). Patients were evaluated on the day before treatment and on the second day after 1 and 2 courses of treatment.

[0071] Serum indicators: blood calcium (Ca), blood phosphorus (P), alkaline phosphatase (ALP); bone metabolism indicators: C-tenninal Cross Linked, β-CTX, procollagen type, P1NP, bone morphogenetic protein (BMP) were analyzed before treatment, after 1 and 2 courses of treatment.

[0072] Bone density measurement (DXA): The dual-energy X-ray absorptiometry instrument (HOLOGIC, USA) was used to measure the bone density of the lumbar spine (L1-L4) and femoral neck at the time of initial diagnosis and on the second day after 1 or 2 courses of medication.

[0073] 4. Efficacy evaluation criteria

[0074] According to the judgment criteria in "Mongolian Medicine Diagnosis and Treatment Methods for 18 Specialties and 49 Diseases" edited by Buren Batu, the degree of improvement of clinical symptoms of the two groups of patients was evaluated.

[0075] The disease score after cure is significantly reduced, and the statistical method is: [score before cure - score after cure] / score before cure × 100%.

[0076] Recovery: Clinical symptoms are significantly improved and symptom scores are significantly reduced to more than 95%;

[0077] Markedly effective: The clinical symptoms significantly improved by more than 70%, and the symptom score decreased by more than 95%;

[0078] Effective: clinical symptoms improved compared with the previous period, with symptom score reduced by 30%≤<70%;

[0079] Ineffective: The patient's clinical symptoms did not improve significantly, and even worsened, and the symptom score decreased by 30%.

[0080] 5. Safety Assessment

[0081] Vital sign testing: electrocardiogram, blood routine, liver and kidney function, coagulation time, quantitative assessment of patients was performed on the day before medication and the day after 1 and 2 courses of treatment.

[0082] Safety assessment criteria:

[0083] Grade I: safe, with no adverse reactions in the subjects;

[0084] Grade II: relatively safe, or with mild adverse reactions, no treatment is required, and medication can continue;

[0085] Level III: The safety factor is relatively high, but there may be negative effects in general situations. It can be used continuously after certain treatment;

[0086] Grade IV: Severe adverse reactions may endanger the life of the subject. The trial should be terminated immediately and active measures should be taken for treatment.

[0087] 6. Statistical processing

[0088] SPSS 26.0 software was used for statistical analysis, and the mean ± standard deviation The measurement data were statistically analyzed and were in accordance with normal distribution. Paired sample t test was used for intra-group comparison, and single sample t test was used for inter-group comparison. Independent sample rank sum test was used for non-normal distribution data. P < 0.05 was considered statistically significant, and P ≥ 0.05 was considered insignificant.

[0089] 3. Research Results

[0090] 1. General data comparison

[0091] This embodiment originally included 90 cases, 45 cases in the control group and 45 cases in the observation group. During the clinical trial, 3 cases in the observation group withdrew from the study due to adverse reactions such as gastrointestinal discomfort and skin itching after taking the medicine, and the follow-up was relieved spontaneously, and 1 case was lost to follow-up; 4 cases in the control group withdrew from the study due to nausea, vomiting, and stomach discomfort, which were relieved spontaneously during follow-up, and 1 case was lost to follow-up. Finally, 41 cases in the observation group and 40 cases in the control group completed the study. The age, menopausal time, body mass index (BMI), and waist circumference of the two groups of cases were compared, and there was no statistically significant difference (P>0.05), and the two groups of patients were comparable.

[0092] 2. Comparison of VAS scores between the two groups before and after treatment

[0093] The VAS scores of the two groups of patients before and after treatment are shown in Table 1. There was no significant difference in the VAS scores of the two groups of patients before treatment (P>0.05). After one course of treatment, the VAS scores of the subjects in the observation group and the control group decreased compared with those before treatment, but the difference between the groups was not large (P>0.05), indicating that both groups of drugs were effective and the difference was not significant. After two courses of treatment, the VAS scores of the observation group and the control group decreased; but the pain relief of the observation group was more obvious than that of the control group (P<0.05). It can be seen that the basic drug plus the pharmaceutical composition of the present invention is superior to the basic drug plus alendronate sodium tablets in the long-term treatment of brittle fractures in relieving pain symptoms. In the intra-group comparison, the numerical difference between the observation group and the control group before treatment was not large; after one and two courses of treatment, it was found that compared with before treatment, the VAS scores of the two groups of patients were reduced and the pain symptoms were significantly relieved. Both groups of drugs were effective in treating brittle fracture pain, and the difference was statistically significant (P<0.05).

[0094] Table 1 VAS scores of the two groups of patients before and after treatment

[0095]

[0096] Note: Inter-group comparison, before treatment, P>0.05, after 1 course of treatment ★ P>0.05, after 2 courses of treatment * P<0.05; intra-group comparison, compared with before treatment # P<0.05, * P<0.05.

[0097] 3. Comparison of ODI index between the two groups before and after treatment

[0098] As shown in Table 2, after treatment, compared with before treatment, the functional impairment of the two groups of patients recovered well. Compared with the control group, after one course of treatment, the functional impairment recovery index of the observation group was better than that of the control group (P<0.05), indicating that the basic drug plus the pharmaceutical composition of the present invention is better than the basic drug plus alendronate sodium tablets in the ODI index of relieving brittle fractures.

[0099] Table 2 ODI index of the two groups of patients before and after treatment (%)

[0100]

[0101] Note: Intra-group comparison, before treatment, P>0.05, after 1 and 2 courses of treatment * P<0.05, # P<0.05; Intergroup comparison, after 1 course of treatment, ★ P<0.05.

[0102] 4. Comparison of Mongolian medicine symptom scores between the two groups before and after treatment

[0103] The total effective rate of Mongolian medicine symptoms was 89.35% in the observation group and 72.40% in the control group. It can be seen that the total effective rate of Mongolian medicine symptoms was better in the observation group than in the control group (Table 3). The clinical symptoms of the single effective rate of Mongolian medicine symptoms in the observation group and the control group were: back pain, indigestion, poor appetite, and insomnia. In terms of the single effective rate of relieving the main and secondary symptoms of Mongolian medicine, the observation group was better than the control group (Table 4). The improvement of Mongolian medicine symptom scores is shown in Table 5. In the intra-group comparison, after one course of treatment, the symptoms of the two groups of patients were significantly improved compared with those before treatment (P<0.05), and there was a difference between the groups (P>0.05). After two courses of treatment, the intra-group comparison of the observation group and the control group showed that the symptoms of the two groups of patients were significantly improved compared with those before treatment (P<0.01). In the inter-group comparison, the Mongolian medicine symptoms of the observation group were significantly relieved compared with those of the control group (P<0.05).

[0104] Table 3 Comparison of the total effective rate of Mongolian medicine symptom score (example, 100%)

[0105]

[0106] Note: Compared with the control group, *P<0.05.

[0107] Table 4 Comparison of the effective rate of Mongolian medicine symptom scores in two groups of patients (example, 100%)

[0108]

[0109] Note: Effective means clinical healing + marked effect + effective. Comparison among the groups: *P<0.05, **P<0.01.

[0110] Table 5 Comparison of Mongolian medicine symptom scores between the two groups of patients before and after treatment

[0111]

[0112] Note: Comparison before and after treatment, comparison within the group * P<0.05, *# P < 0.05; ** P<0.01, **## P<0.01, comparison between groups *# P>0.05, **## P<0.01.

[0113] 5. Comparison of quantitative scores of spleen and stomach discomfort, soreness of waist and knees, difficulty in walking, cramps in lower limbs, and difficulty in carrying weight between the two groups

[0114] The comparison of spleen and stomach function, muscle strength, difficulty in walking, cramps in lower limbs, and difficulty in holding weight between the two groups of patients is shown in Table 6. As shown in Table 6, compared with the control group, all indicators in the observation group were significantly improved after treatment (P<0.05).

[0115] Table 6 Comparison of spleen and stomach function, muscle strength, difficulty walking, lower limb cramps, and difficulty holding weight between the two groups of patients

[0116]

[0117] Note: Intra-group comparison, before treatment, P>0.05, after 1 course of treatment, P<0.05, after 2 courses of treatment, P<0.01; inter-group comparison, before treatment, P>0.05, after 1 course of treatment, *P>0.05, after 2 courses of treatment, **P<0.05.

[0118] 6. Comparison of quantitative scores of MH, SF, RE, VT, GH, BP, RP, and PF between the two groups

[0119] The comparison of mental health (MH), social function (SF), emotional role (RE), energy (VT), general health (GH), bodily pain (BP), physiological role (RP), and physiological function (PF) between the two groups of patients is shown in Table 7. Compared with the control group, the MH, SF, RE, VT, GH, BP, RP, and PF indicators of the observation group were better than those of the control group after treatment (P<0.05).

[0120] Table 7 Comparison of MH, SF, RE, VT, GH, BP, RP, and PF indices between the two groups

[0121]

[0122]

[0123] Note: Intra-group comparison: P>0.05 before treatment, *P<0.05, #P<0.05 after 1 course of treatment, **P<0.01, ##P<0.01 after 2 courses of treatment; Inter-group comparison: P>0.05 before treatment, P>0.05 after 1 course of treatment, P<0.05 after 2 courses of treatment.

[0124] 7. Comparison of bone density values ​​between the two groups before and after treatment

[0125] The bone density of the lumbar spine and femoral neck of the two groups of cases before and after treatment is shown in Table 8. After one course of treatment, there was no significant change in the bone density of the lumbar spine and femoral neck in the observation group and the control group (P>0.05). After two courses of treatment, the bone density value showed an upward trend compared with that before treatment, but there was no significant difference; there was no significant difference between the two groups in the comparison between the two groups (P>0.05).

[0126] Table 8 Bone density of lumbar spine and femoral neck before treatment, after 1 course of treatment and after 2 courses of treatment in the two groups

[0127]

[0128] Note: Before and after treatment, comparison within and between groups: *P>0.05, #P>0.05.

[0129] 8. Comparison of serum indicators between the two groups

[0130] The comparison results of serum indicators between the two groups are shown in Table 9. After 1 and 2 courses of treatment, there was no significant difference in blood calcium and blood phosphorus levels between the observation group and the control group (P>0.05). The alkaline phosphatase levels of the two groups increased after treatment (P<0.05). Compared with the other groups, the alkaline phosphatase levels of the observation group were higher than those of the control group after 1 course of treatment and 2 courses of treatment, respectively, and the difference was significant (P<0.05).

[0131] Table 9 Changes of blood calcium, blood phosphorus and ALP levels in the two groups before and after treatment

[0132]

[0133] Note: Intra-group comparison, before and after treatment for 1 and 2 courses, * P<0.05, ** P>0.05; Inter-group comparison, before treatment, after 1 and 2 courses of treatment, P>0.05. Intra-group and inter-group comparison, before and after treatment, # P>0.05. Intra-group and inter-group comparison, before and after treatment, ★ P<0.05, ★★ P<0.05.

[0134] 9. Comparison of bone metabolism markers between the two groups

[0135] The changes of type I collagen C-terminal peptide (β-CTX) and type I procollagen amino-terminal propeptide (P1NP) in the two groups before and after treatment are shown in Table 10. After 2 courses of treatment, β-CTX in both groups decreased significantly, and the value in the observation group was significantly lower than that in the control group (P<0.01); after 2 courses of treatment, P1NP in both groups increased significantly, and the value in the observation group was significantly better than that in the control group (P<0.05).

[0136] Table 10 Comparison of changes in β-CTX and P1NP between the two groups before treatment, after one course of treatment, and after two courses of treatment

[0137]

[0138] Note: Before treatment, the comparison between the two groups was P>0.05; after one course of treatment, the comparison between and within the two groups was P>0.05; after two courses of treatment, * indicates within the group, # indicates between the two groups, *P<0.05, #P<0.05.

[0139] 10. Safety evaluation

[0140] During the trial, both groups of patients experienced adverse reactions such as gastrointestinal discomfort and urticaria, which were relieved on their own during follow-up. No abnormalities were found in other living conditions, electrocardiograms, blood tests, urine tests, liver and kidney functions, and coagulation tests. The peripheral blood Cr, BUN, TG, and ALT index data of the two groups of cases were further evaluated. The results showed that there was no significant difference in the scores of Cr, BUN, TG, and ALT before treatment, after one course of treatment, and after two courses of treatment, and they were all within the normal range.

[0141] This example demonstrates that the pharmaceutical composition of the present invention has a significant therapeutic effect on the treatment of brittle fractures in clinical practice.

[0142] Example 2 Animal Experimental Study on the Effect of the Pharmaceutical Composition on Bone Microstructure and Bone Metabolism in Rats with Fragile Fracture

[0143] 1. Experimental Materials

[0144] Animals: A total of 60 12-month-old SPF female SD rats weighing (280±20) g; a total of 25 1-day-old SPF SD rats weighing (10±5) g were provided by Nanjing Jiangning District Qinglongshan Animal Experiment Center. Ethics number A200904.

[0145] Drug: The drug composition and alendronate sodium tablets were the same as in Example 1, and were prepared into drug solutions of different concentrations.

[0146] 2. Experimental Methods

[0147] 1. Experimental Grouping

[0148] After one week of adaptive feeding, the rats were divided into sham operation group (Sham), model group (Ovx), low-dose group (Low) of drug combination, medium-dose group (Midde), high-dose group (High), and alendronate sodium tablets group (AST) according to the random number table method, with 10 rats in each group.

[0149] 2. Model preparation

[0150] After the osteoporosis rat model was established by castration, lumbar compression fracture was performed by three-point bending test. The rats were anesthetized by isoflurane inhalation, skin was prepared, and disinfected. A dorsal midline incision was made to expose the abdominal cavity layer by layer, the ovaries were found, and the bilateral ovarian tissues were ligated and removed, and sutured layer by layer. The sham operation group retained the ovaries, and the fat tissue of equal mass around the ovaries was removed and sutured layer by layer. The whole process was sterile, and penicillin ointment was applied to the wound within 3 days after surgery to prevent wound infection.

[0151] 3. Drug intervention

[0152] According to the conversion method of body surface area between rats and humans, the drug combination groups were given oral administration of low, medium and high doses (7.5 g·kg -1 , 15g·kg -1 , 30g·kg -1 )2mL·d -1 The positive control group was treated with alendronate sodium tablets (15 g kg -1 )2mL·d -1 Rats in the sham operation group and the model group were gavaged with the same dose of saline (15 g kg -1 )2mL·d -1 The dosing period was 12 weeks.

[0153] 4. Sample collection

[0154] After 12 weeks of continuous administration, the rats were anesthetized by isoflurane inhalation, and blood was collected from the abdominal aorta. The blood was centrifuged at 3500rpm for 10min, and the upper serum was collected and stored at -20℃. The femur specimens from both sides of the rats in each group were collected, of which the femurs of 5 rats were fixed in 4% paraformaldehyde, and the rest were frozen with liquid nitrogen and stored at -80℃.

[0155] 5. Index detection

[0156] Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of TRAP and ALP in serum; polarography was used to detect serum trace elements, and Micro-CT was used to detect changes in femoral microstructure; HE staining was used to observe changes in femoral pathological morphology; immunohistochemistry was used to detect the protein expressions of Runx2 and OCN in bone tissue; WB was used to detect the protein expressions of osteoblast-related proteins BMP6, BMP2, Wnt1, β-catenin, and OCN in bone tissue; the biomechanical properties of bones were detected; the mitochondrial membrane potential level of bone tissue was detected, and the NAD in liver tissue was detected. + / NADH、Na + -K + -ATPase levels.

[0157] 3. Experimental Results

[0158] 1. Micro-CT test results

[0159] Five rats were randomly selected from each group, and the Micro-CT test results showed that ( Figure 1 ): Compared with the Sham group, the trabecular bone density in the Ovx group was significantly reduced; sagittal images also showed that there was cortical bone loss, thinning of the trabecular bone structure, and larger gaps; trabecular bone was almost invisible in the 2D plane image. Compared with the Ovx group, the trabecular bone density in the low, medium, and high dose groups (Low, Midde, High) of the drug combination and the positive control group (AST) was significantly increased, and the bone gaps were relatively smaller, indicating that the drug combination and alendronate sodium tablets can prevent the loss of trabecular bone minerals caused by Ovx; there was no significant difference between the drug combination group and the AST group.

[0160] The topological correlation data of bone microstructure showed that ( Figure 2 ), the four bone parameters of BMD, BV / TV, Tb.Th and Tb.N in the Ovx group were significantly lower than those in the Sham group; the drug combination and alendronate sodium tablets could improve these four parameters to varying degrees, among which BMD, BV / TV, Tb.Th and Tb.N were significantly improved compared with the Ovx group; compared with the Sham group, the Tb.Sp in the Ovx group was significantly increased, and the difference was significant; compared with the Ovx group, the Tb.Sp in the drug combination group and the AST group was significantly reduced, and the difference was obvious; there was no obvious difference between the different dose groups of the drug combination, and there was no statistical significance, and there was no obvious difference between the drug combination group and the AST group; but there was still a certain gap compared with the Sham group.

[0161] 2. Bone biomechanical test results

[0162] After intervention with the drug combination, the biomechanical properties of the rat femur changed significantly ( Figure 3 ). After 12 weeks of administration, compared with the Sham group, the maximum load of the femur of rats in the Ovx group was significantly reduced, and the difference was statistically significant (P < 0.01). The drug combination can change this trend. Compared with the Ovx group, the maximum load of the femur of rats in the medium and high dose groups of the drug combination group was significantly improved, and the difference was statistically significant (P < 0.01). Among them, the effect of the medium dose group was the most prominent. Compared with the Ovx group, the maximum displacement of the Sham group was greatly improved, and the difference was statistically significant (P < 0.05), and the change trend of the drug combination was also obvious, and the difference was statistically significant (P < 0.05). Compared with the Sham group and the Ovx group, the difference in maximum stiffness was significant and statistically significant (P > 0.01); compared with the Ovx group, the drug combination group had an improvement trend, but the difference was not statistically significant (P > 0.05).

[0163] 3. Results of bone tissue pathological morphology observation (HE staining)

[0164] The results of distal femoral tissue pathological staining showed that ( Figure 4 ), the trabeculae of rats in the Sham group were closely arranged, with clear structure, small bone marrow cavity and more bone; the trabeculae in the Ovx group were obviously broken, with disordered structure, dense bone marrow cavity and scarce bone. Compared with the Ovx group, the trabeculae in the drug combination (Low, Middle, High) group and the alendronate sodium tablets (AST) group were less broken, arranged more closely, with fewer cavities and significantly increased bone content; among them, the drug combination (Middle, High) group could significantly improve the pathological changes of trabecular tissue.

[0165] 4. Expression of serum bone metabolism indicators ALP and TRAP

[0166] The serum of rats was collected and tested by ELISA for related indicators. Figure 5 As shown. The results showed that compared with the Sham group, the ALP level in the serum of rats in the Ovx group decreased (P < 0.05), and the TRAP level increased (P < 0.01). Compared with the Ovx group, the TRAP level in the low-dose group of the drug combination decreased, and the difference was statistically significant (P < 0.05). The TRAP level in the drug combination (Middle, High) group and the AST group decreased more significantly, with significant statistical significance (P < 0.01). In addition, compared with Ovx, the drug combination group (High) and the AST group can significantly increase the ALP level, and the difference is statistically significant (P < 0.05).

[0167] 5. Contents of trace elements Zn, Ca and Na in serum

[0168] The results of serum trace element detection showed that compared with the Sham group, the Zn content in the serum of rats in the Ovx group was significantly decreased (P < 0.05), and the Zn content in the serum of the drug combination (Middle, High) group and the AST group was significantly increased (P < 0.01); compared with the Sham group, the levels of Ca and Na in the Ovx group were decreased, and the drug combination intervention could effectively improve the Ca level (P < 0.05).

[0169] 6. Changes in the levels of osteogenic-related proteins BMP6, BMP2, Wnt1, and β-catenin

[0170] Western blot was used to detect the expression levels of BMP6, BMP2, Wnt1 and β-catenin proteins in the femoral tissues of each group. Figure 6As shown in Table 11. Compared with the Sham group, the levels of Wnt1 and β-catenin proteins in the Ovx group were significantly reduced (P < 0.01); compared with the Ovx group, the concentrations of Wnt1 and β-catenin proteins in the drug combination group and the AST group were significantly increased, and the difference was statistically significant (P < 0.05), and the drug combination group also increased with the increase of drug dose, but the difference was not statistically significant (P > 0.05). Compared with the Sham group, the levels of BMP6 and BMP2 proteins in the Ovx group were significantly reduced (P < 0.01); compared with the Ovx group, the levels of BMP6 and BMP2 proteins in the drug combination group and the AST group were increased, and the difference was statistically significant (P < 0.05). The drug combination groups showed an upward trend with the increase of drug dose, but the difference was not statistically significant (P > 0.05). Comparison among the groups showed that the levels of BMP6, BMP2, Wnt1, and β-catenin proteins in the drug combination group were higher than those in the AST group, but without statistical significance (P>0.05).

[0171] Table 11 BMP6, BMP2, wnt1, β-catenin protein expression results

[0172]

[0173] Note: Compared with the Ovx group, P<0.05, P<0.01.

[0174] 7. Protein expression levels of osteogenic-related proteins Runx2 and OCN in bone tissue

[0175] The immunohistochemical results of Runx2 in bone tissue showed that ( Figure 7 ), the positive expression of Runx2 in bone tissue can be seen in the bone marrow, cells, and trabeculae, and the normal group is expressed in light yellow, light brown, and dark brown. A small amount of positive reaction can be observed in the Ovx group. The three groups intervened with the drug combination showed light yellow and dark yellow positive reactions in the bone marrow and cells; compared with the Ovx group, the color of the drug combination group increased significantly, but was still lower than that of the Sham group and the AST group (P<0.05).

[0176] The immunohistochemical results of OCN in bone tissue showed that ( Figure 8 ), compared with the Sham group, the number of OCN-positive osteoblasts in the Ovx group decreased; after the intervention of the drug combination, the number of OCN-positive osteoblasts increased significantly, and the difference was statistically significant compared with the Ovx group (P<0.05). Compared with the AST group, the color of the drug combination group was darker yellow, but there was no statistical significance.

[0177] 8. Detection of mitochondrial membrane potential level in bone tissue

[0178] The results of bone tissue mitochondrial membrane potential level detection are as follows Fig. 9 Compared with the Sham group, the bright red fluorescence ratio of the Ovx group was significantly reduced, while the cyan fluorescence ratio was significantly increased, indicating that the mitochondrial membrane power supply level decreased. Compared with the Ovx group, the bright red fluorescence ratio of the drug combination group was significantly increased, while the cyan fluorescence ratio was relatively reduced. This shows that the drug combination can effectively increase the mitochondrial membrane potential level and improve mitochondrial function.

[0179] 9. Comparison of liver energy metabolism indicators in rats of each group

[0180] Compared with Sham, the liver NAD of rats in Ovx group + Compared with Ovx, the liver NAD of rats in the medium and high dose groups of the drug composition decreased (P < 0.05). + / NADH increased significantly, and the difference was statistically significant (P<0.05, Fig.10 In addition, compared with the Sham group, the Na + -K + -ATPase activity was significantly decreased (P<0.05); compared with the Ovx group, the Na + -K + - ATPase activity increased, especially Na in the high-dose group of the drug composition + -K + -ATPase activity was significantly increased, and the difference was statistically significant (P<0.05); compared with the other groups, it was lower than that of the AST group, but the difference was not statistically significant ( Fig.10 B).

[0181] This example demonstrates that the pharmaceutical composition of the present invention can improve the bone microstructure of rats, regulate bone metabolism, and thus play a role in treating brittle fractures.

[0182] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A pharmaceutical composition for treating brittle fractures, characterized in that: The raw materials include the following components in parts by weight: 5-20 parts of pomegranate, 5-10 parts of tribulus terrestris, 10-15 parts of piper longum, 2-20 parts of alpinia oxyphylla, 5-15 parts of okra fruit, 5-15 parts of cinnamon bark, 5-15 parts of polygonatum, 5-20 parts of safflower, 5-15 parts of asparagus, 5-15 parts of polygonatum and 5-20 parts of trichosanthes.

2. The pharmaceutical composition according to claim 1, characterized in that The raw materials include the following components by weight: 10 parts of pomegranate, 5 parts of tribulus terrestris, 15 parts of piper longum, 20 parts of alpinia oxyphylla, 5 parts of winter mallow fruit, 10 parts of cinnamon bark, 10 parts of polygonatum, 20 parts of safflower, 5 parts of asparagus, 10 parts of polygonatum and 5 parts of trichosanthes.

3. A method for preparing the pharmaceutical composition according to claim 1 or 2, characterized in that: The method comprises the steps of mixing the raw materials by weight, boiling with water and then concentrating to obtain the pharmaceutical composition.

4. The preparation method according to claim 3, characterized in that: The amount of water added is 10 times the weight of the mixed raw materials.

5. The preparation method according to claim 3, characterized in that: The decoction time is 45 minutes.

6. The preparation method according to claim 3, characterized in that: After decoction, it is concentrated to 15% to 35% of the original volume.

7. Use of the pharmaceutical composition according to claim 1 or 2 in the preparation of a drug for treating brittle fractures.

8. Use of the pharmaceutical composition according to claim 1 or 2 in the preparation of a drug for improving osteoporosis.

9. Use of the pharmaceutical composition according to claim 1 or 2 in the preparation of a drug for regulating bone metabolic balance.

10. The use according to any one of claims 7 to 9, characterized in that: The dosage forms of the drug include pills, powders, tablets and granules.

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