CN112891366B - Traditional Chinese medicine active ingredient formula for treating postmenopausal osteoporosis and application thereof - Google Patents

Traditional Chinese medicine active ingredient formula for treating postmenopausal osteoporosis and application thereof Download PDF

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CN112891366B
CN112891366B CN201911221261.5A CN201911221261A CN112891366B CN 112891366 B CN112891366 B CN 112891366B CN 201911221261 A CN201911221261 A CN 201911221261A CN 112891366 B CN112891366 B CN 112891366B
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朱晓峰
张荣华
杨丽
孙珂焕
梅雯惠
辛灵
崔琰
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Abstract

The invention provides a traditional Chinese medicine active ingredient formula for treating postmenopausal osteoporosis and application thereof, belonging to the field of medicines. The Chinese medicinal active ingredient composition comprises Chinese medicinal active ingredients of lycium barbarum polysaccharide, icariin, polydatin, quercetin and salvianic acid A sodium in a weight ratio (mg) of (50-350): 6-100): 50-180): 1-10): 10-100. The traditional Chinese medicine active ingredients of the invention have reasonable formula compatibility, supplement each other, definite curative effect and strong innovation, can be effectively applied to the treatment of postmenopausal osteoporosis, and repeatedly verified, the formula can obviously improve the bone density of postmenopausal osteoporosis model animals, improve the bone microstructure, is safe and effective, and can be used as a long-term medicine for postmenopausal osteoporosis. The Chinese medicinal composition can be further prepared into a medicinal preparation, has small dosage form, is convenient to carry and take, can obtain better economic and social benefits, and has wide application prospect.

Description

Traditional Chinese medicine active ingredient formula for treating postmenopausal osteoporosis and application thereof
Technical Field
The invention belongs to the field of medicines, and particularly relates to a traditional Chinese medicine active ingredient formula for treating postmenopausal osteoporosis and application thereof.
Background
Postmenopausal osteoporosis is a public health problem seriously threatening the health of middle-aged and elderly people, and about 2 hundred million patients are worldwide. In women over 60 years old in our country, the prevalence of osteoporosis is as high as 40% -50%, of which about 30% -50% will experience osteoporosis related fractures, which will very seriously affect the health and quality of life of the elderly, leading to shortened lifespan. In China, according to statistics, the number of patients with osteoporosis and related fracture exceeds 9000 ten thousand, the annual medical expense exceeds 250 hundred million yuan, and the financial and manpower burden of the country and families is increased seriously (Chinese white paper for osteoporosis, 2009). Although the varieties of the medicines for treating osteoporosis in the current market are various, most medicines for treating osteoporosis still have the defects of poor safety, great side effect, incapability of effectively reducing fracture risk and the like.
The main reason of postmenopausal osteoporosis is estrogen deficiency, which occurs in female patients, and multiple factors such as heredity, life style and nutrition are involved in the onset of osteoporosis, and the family history of osteoporosis, women with special genes influencing bone mass, calcium deficiency, lack of physical activity, a large amount of smoking and drinking, and early menopause or premenopausal bilateral ovariectomy are all high-risk factors. At present, the medicines for treating osteoporosis mainly comprise basic supplements, medicines for inhibiting bone resorption and medicines for promoting bone formation. The basic supplement mainly comprises calcium and vitamin D. The medicines for clinically inhibiting the bone resorption of osteoclast mainly comprise diphosphate, estrogen and receptor regulator thereof, calcitonin and the like. The medicines for promoting bone formation mainly comprise parathyroid hormone, strontium salt medicines, vitamin K2 and the like. However, the toxic side effects of drugs limit long-term use, e.g. estrogens and their receptor modulators may increase the risk of causing breast cancer and uterine bleeding and venous thrombosis; bisphosphonates can cause side effects of mandibular necrosis and atypical femoral fractures. Therefore, the search for effective therapeutic agents remains a problem that research is urgently needed at present.
Osteoporosis belongs to the category of 'bone atrophy', 'bone rheumatism' and the like in traditional Chinese medicine. The traditional Chinese medicine holds that the kidney governs bones and produces marrow and stores essence, which indicates that the growth and development of human skeleton depend on kidney essence. Especially, after menopause, the deficiency of kidney essence is often caused by the preponderance and the decline of kidney essence, which leads to the empty deficiency of bone marrow, the bone can not be nourished, the malnutrition of the bone can be caused, and in addition, the generation of osteoporosis is aggravated along with the generation of blood stasis, the bone nutrition disorder is caused by the blood stasis along with the growth of age. The treatment is performed by taking kidney tonifying, bone strengthening and blood circulation activating as methods, and various traditional Chinese medicine formulas are applied to treatment, but the defects of inconvenience in carrying and taking, undefined components and the like exist.
Disclosure of Invention
Aiming at the above contents, in order to make up the defects of the existing medicine for treating postmenopausal osteoporosis, the invention mainly aims to provide a traditional Chinese medicine active ingredient formula for treating postmenopausal osteoporosis.
The invention also aims to provide application of the traditional Chinese medicine active ingredient formula for treating postmenopausal osteoporosis.
The purpose of the invention is realized by the following technical scheme:
a traditional Chinese medicine active ingredient formula for treating postmenopausal osteoporosis comprises the following traditional Chinese medicine active ingredients in parts by weight: icariin: polydatin: and (3) quercetin: the sodium danshensu comprises (50-350): (6-100): (50-180): (1-10): (10-100): the unit of measurement is mg, and the measurement interval is the minimum dosage and the maximum dosage.
The traditional Chinese medicine active ingredient formula for treating postmenopausal osteoporosis is preferably any one group calculated according to the weight ratio as follows:
(1) wolfberry polysaccharide: icariin: polydatin: and (3) quercetin: sodium danshensu 50:6:50:1: 10;
(2) wolfberry polysaccharide: icariin: polydatin: and (3) quercetin: sodium danshensu 350:50:180:10: 100;
(3) wolfberry polysaccharide: icariin: polydatin: and (3) quercetin: sodium danshensu 200:30:120:5: 60;
(4) wolfberry polysaccharide: icariin: polydatin: and (3) quercetin: sodium danshensu 50:100:120:1: 100;
(5) wolfberry polysaccharide: icariin: polydatin: and (3) quercetin: sodium danshensu 80:15:80:3: 20;
(6) wolfberry polysaccharide: icariin: polydatin: and (3) quercetin: danshensu sodium (200: 30:160:8: 80).
Preferably, the purity of the lycium barbarum polysaccharide in the traditional Chinese medicine active ingredient formula for treating postmenopausal osteoporosis is over 90% determined by high performance liquid chromatography (HLPC method); the icariin, polydatin, quercetin and salvianic acid A sodium are respectively determined by an HLPC method, and the content of the icariin, the polydatin, the quercetin and the salvianic acid A sodium is not less than 98 percent.
The traditional Chinese medicine active ingredient formula for treating postmenopausal osteoporosis is applied to preparation of a medicine for treating postmenopausal osteoporosis.
The medicine for treating postmenopausal osteoporosis also contains one or more pharmaceutically acceptable auxiliary materials.
The auxiliary material is preferably at least one of excipient, sustained release agent, filler, adhesive, wetting agent, disintegrating agent, absorption enhancer, surfactant, antibacterial agent, aromatic agent, antioxidant, pH regulator, protective agent, diluent, lubricant, solvent, matrix or carrier material.
The traditional Chinese medicine active ingredient formula for treating postmenopausal osteoporosis can be prepared into various dosage forms by adopting the conventional method in the field, such as granules, tablets, pills, capsules or oral liquid and other oral preparations.
The Chinese medicinal composition selects active ingredients of Chinese medicaments for tonifying kidney, strengthening bones, promoting blood circulation and removing blood stasis, and the Chinese medicinal composition consists of lycium barbarum polysaccharide, icariin, polygonin, quercetin and danshensu sodium. Icariin has effects of improving gonadal function, regulating organism immunity, promoting bone growth, delaying aging, protecting heart and cerebral vessels system, promoting osteoblast differentiation, and inhibiting osteoclast differentiation. The polydatin is an active ingredient of traditional Chinese medicine giant knotweed, has the effects of improving microcirculation, resisting oxidation, reducing blood fat, resisting lipid peroxidation and the like, and also has good effect of promoting osteoblast proliferation and differentiation. Quercetin is present in various plants and traditional Chinese medicines, has good effects of eliminating phlegm and relieving cough, also has effects of lowering blood pressure, reducing blood lipid, increasing coronary blood flow, etc., and has strong effect of promoting bone cell differentiation. The salvianic acid A sodium is sodium salt of salvianic acid A as active component of Saviae Miltiorrhizae radix, has good chemical stability compared with salvianic acid A, has effects of inhibiting platelet aggregation, resisting bacteria and inflammation, enhancing organism immunity, resisting atherosclerosis, reducing blood lipid, increasing bone mass of castrated rat, and inhibiting osteoclast differentiation. The five traditional Chinese medicine active ingredient combination formula not only accords with the principle of tonifying kidney and activating blood to treat postmenopausal osteoporosis in traditional Chinese medicine, but also accords with the pharmacological mechanism of modern medicine, experiments show that the formula can improve the bone density of ovariectomized rats, improve the bone microstructure and the like. The method has better social and economic significance for further research and development.
The brief introduction of the active ingredients of the traditional Chinese medicine is as follows:
1. wolfberry polysaccharide (Lycium barbarum polysaccharides) which is soluble in water and proteoglycan with molecular weight of 22-25 KD; polysaccharide side chains consisting of six hexoses (rhamnose, arabinose, xylose, mannose, galactose and glucose) and a protein main chain consisting of 18 amino acids are connected in a Glycan-O-Der mode through beta-glycosidic bonds or alpha-pyranose/furanose.
2. Icariin (Icraiin), molecular formula: c 33 H 40 O 15 (ii) a Molecular weight: 676.6617, respectively; CAS number: 56692-02-5; chemical structural formula:
Figure BDA0002300918730000031
3. polydatin (Polydatin), molecular formula: c 20 H 22 O 8 (ii) a Molecular weight: 390.40, respectively; CAS number: 27208-80-6; chemical structural formula:
Figure BDA0002300918730000041
4. quercetin (Quercetin), molecular formula: c 15 H 10 O 7 (ii) a Molecular weight: 302.23, respectively; CAS number: 117-39-5; chemical structural formula:
Figure BDA0002300918730000042
5. salvianic acid A sodium (Salvianic acid A sodium), molecular formula: c 9 H 9 NaO 5 (ii) a Molecular weight: 220.16, respectively; CAS number: 67920-52-9; chemical structural formula:
Figure BDA0002300918730000043
compared with the prior art, the invention has the following advantages and effects:
the traditional Chinese medicine composition has rich raw materials and convenient and fast preparation method, can be effectively used for treating postmenopausal osteoporosis, can be developed into a new medicine for treating the postmenopausal osteoporosis, has higher economic and social benefits, and is an innovation in traditional Chinese medicines.
Drawings
FIG. 1 is a photograph showing the results of bone trabecular HE staining of femoral bone tissue in rats of each group.
FIG. 2 is a diagram of the results of micro-CT of the trabecular bone of femur of each group of rats.
FIG. 3 is a graph of the lumbar spine micro-CT detection results of rats in each group.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
In the specific implementation of the invention, the active ingredient component formula of the traditional Chinese medicine consists of 50mg of lycium barbarum polysaccharide, 6mg of icariin, 50mg of polydatin, 1mg of quercetin and 10mg of danshensu sodium.
Example 2
In the specific implementation of the invention, the active ingredient component formula of the traditional Chinese medicine consists of 350mg of lycium barbarum polysaccharide, 50mg of icariin, 180mg of polydatin, 10mg of quercetin and 100mg of danshensu sodium.
Example 3
In the specific implementation of the invention, the active ingredient components of the traditional Chinese medicine comprise 200mg of lycium barbarum polysaccharide, 30mg of icariin, 120mg of polydatin, 5mg of quercetin and 60mg of danshensu sodium.
Example 4
In the specific implementation of the invention, the active ingredient components of the traditional Chinese medicine comprise 50mg of lycium barbarum polysaccharide, 100mg of icariin, 120mg of polydatin, 1mg of quercetin and 100mg of danshensu sodium.
Example 5
In the specific implementation of the invention, the active ingredient component formula of the traditional Chinese medicine consists of 80mg of lycium barbarum polysaccharide, 15mg of icariin, 80mg of polydatin, 3mg of quercetin and 20mg of danshensu sodium.
Example 6
In the specific implementation of the invention, the active ingredient components of the traditional Chinese medicine comprise 200mg of lycium barbarum polysaccharide, 30mg of icariin, 160mg of polydatin, 8mg of quercetin and 80mg of danshensu sodium.
Example 7
The traditional Chinese medicine component formula described in any one of embodiments 1 to 6 is mixed uniformly, and auxiliary materials acceptable in pharmaceutical preparations are added according to a certain proportion, and the mixture is prepared into capsules, tablets, pills, granules or oral liquid according to a conventional preparation method.
The purity of the lycium barbarum polysaccharide in the embodiment is over 90% through high performance liquid chromatography (HLPC method); the icariin, polydatin, quercetin and salvianic acid A sodium are respectively determined by an HLPC method, and the content of the icariin, the polydatin, the quercetin and the salvianic acid A sodium is not less than 98 percent.
Effect example 1 study on therapeutic Effect of drugs
The method is effective and feasible, has rich raw materials and has better practical application value. The traditional Chinese medicine active ingredient formula for treating postmenopausal osteoporosis has the effects of tonifying kidney, strengthening bones and promoting blood circulation, and is effectively used for treating postmenopausal osteoporosis. The invention repeatedly researches the curative effect of the prescription obtained in each embodiment on the postmenopausal osteoporosis of the rat model, and obtains satisfactory curative effect, and the research data is as follows:
1. experimental Material
1.1 animals: 72 female SPF-grade SD rats are 6 months old and 380 + -20 g in weight (animal qualification number: SCXK (Guangdong) 2013-.
1.2 medicine: firstly, a callqi D tablet (0.1 g/tablet, Huishi-Baigong pharmacy Co., Ltd., USA) is ground into fine powder before use, and is added with physiological saline to prepare 1.0mg/mL suspension; ② the active ingredients of the traditional Chinese medicine formula: lycium barbarum polysaccharides are purchased from Beijing Sorbao science and technology Limited (SP9311, Beijing), icariin (110737, Beijing), polydatin (111575, Beijing), quercetin (100081, Beijing), and sodium danshensu (110855, Beijing) from Chinese food and drug testing institute.
1.3 Main instruments: prodigy type dual-energy X-ray bone densitometer: GE corporation, usa; Micro-CT appearance: ZKKS-Sharp-MCT. Hard tissue microtomes, Leica model 2500, germany. Leica QwinV3.2 image analysis System, Germany.
2. Experimental methods
2.1 model preparation
Rats are fasted for 12 hours before operation without water prohibition, weighed before operation and then injected with 2% sodium pentobarbital (0.2mL/100g) for general anesthesia in the abdominal cavity, and after the rats are anesthetized and lie still, the rats are fixed on a plate with the abdominal surface facing upwards and the supine position. Shaving hair at an opening area of a lower abdominal center operation by using a hair shaving cutter, paving an operation hole towel, disinfecting by iodophor and alcohol, taking the lower abdominal center of a rat, longitudinally cutting for 2-3 cm along a white abdominal line, opening an abdominal cavity, separating muscle layers and connective tissues layer by layer, exposing ovaries at two sides, wherein the ovaries are deep pink granular tissues which are mostly covered by surrounding adipose tissues, tying surrounding connected blood vessels and oviducts and other tissues by silk after being lifted, cutting the ovaries, suturing the abdominal membrane layers, the muscle layers and the cortex to close the abdomen layer by layer, and disinfecting abdominal wounds by 75% medical alcohol. To prevent infection, one dose of penicillin was injected intraperitoneally (8 ten thousand units/mL in sterile saline). The sham group removed a small amount of fat after the abdomen was opened and retained the ovaries, and the other treatments were as before.
2.2 grouping and administration
Rats were randomly divided into a sham operation group, a model group, a formula low dose group, a formula medium dose group, a formula high dose group, and a calqi group D, with 12 rats per group. Performing normal saline intragastric administration on a sham operation group and a model group after 12 weeks and 13 weeks of molding; the low, medium and high dosage groups are prepared by 36.0, 72.0 and 114.0mg/kg/d gavage of Chinese medicinal active formula (example 1 formula), and are prepared into suspension with physiological saline before use; in the calqi group D, gavage was performed 1 time per day by using calqi D (0.1g/kg/D), and the material was obtained after 24 weeks.
2.3 obtaining materials and determining indexes
2.3.1 general case: the rat was observed for changes in the mental status, activity, fur, secretions, intake and intake of water, stool and urine, and body weight.
2.3.2 measurement of bone Density: respectively in the 4 th week, the 8 th week and the 12 th week of intervention of each group of rats, randomly extracting 4 rats in each group, injecting 2% pentobarbital sodium into the abdominal cavity for anesthesia, flatly spreading the rats on a scanning plate with the abdominal surface facing downwards, unfolding four limbs, starting a detection scan by a dual-energy X-ray bone density analyzer (DXA), obtaining a bone density scanning image, and measuring the bone density (BMD) of the whole body, the head, the 4 th, the 5 th lumbar vertebra, the thighbone, the proximal femur, the distal femur and the humerus of each group of rats. Data were analyzed using small animal BMD software to compare bone density at various sites in different periods for each group of rats.
2.3.3 detection of trabecular bone microstructure: after 24 weeks, all rats were sacrificed by ether anesthesia and heart blood sampling, and fresh bone tissue was fixed and embedded to make bone slices. Bone tissue HE staining for observation of bone trabecular structure:
(1) decalcification of bone tissue, taking femoral and lumbar vertebra specimens, dehydrating by an automatic dehydrator, probing the bone tissue by a disposable syringe needle until the bone tissue can be punctured easily, taking out, and putting into water flow for showering for 12 h.
(2) Bone tissue slicing: taking out femur and lumbar vertebra tissue, and washing with running water for 30 min; soaking in 95% ethanol I and 95% ethanol II for 2 hr respectively; sequentially immersing in absolute ethyl alcohol I, II and III for 1h, 1h and 0.5 h; sequentially immersing in dimethylbenzene I, II and III for 1h, 1h and 0.5 h; treating the paraffin I, II and III for 0.5h, 1h and 0.5h respectively; embedding by an embedding machine; slicing with a rotary microtome, selecting continuous slices with a thickness of about 3 μm at the distal metaphysis, sticking on an anti-slip glass slide, and baking at 65 deg.C for at least 1 h. (3) Dyeing: taking out the dried slices, soaking the slices in dimethylbenzene I and dimethylbenzene II for 5min respectively, and dewaxing; immersing in anhydrous alcohol I, II for 1min respectively; then treated with 95%, 80%, 70% ethanol in the following order: 1min, 1min and 5min, and 1min with distilled water; the sections were stained with hematoxylin for 10 min; flushing with running water for 1 min; l% hydrochloric acid 3 s; flushing with running water for 12 min; washing with distilled water for 2 min; eosin staining for 20 s; washing with running water for 3 s; 80% ethanol, 30 s; 90 percent and 95 percent ethanol for 1min respectively; soaking in anhydrous ethanol I and II for 3min respectively; immersing slices in xylene I for 1 min; xylene II, 1 min; xylene III, transparent in 1 min; and (5) sealing the neutral gum.
The morphological changes of the microstructure of the femoral bone tissue, including the changes of the number of trabeculae, the thickness of the trabeculae, the gap of the trabeculae, the size of a medullary cavity, the number of trabecular connection points and the number of free tail ends, and the like, are observed by using an optical microscope. The cancellous bone in the cavity above the proximal tibial growth plate of the rat was assayed and analyzed by using a Leica DMLA full-automatic microscope and a Leica QwinV3.2 full-automatic image analysis system. The parameters, units and measurement methods directly measured by the full-automatic image analysis system are shown in table 1, and then the parameters directly measured are calculated by formulas (the calculation formulas are shown in table 2) for analysis.
TABLE 1 name and Unit of parameters of trabecular bone
Figure BDA0002300918730000081
The calculated parameters are as follows:
trabecular bone area percentage: refers to the percentage of bone trabecula occupying the area of bone tissue, reflecting the amount of bone mass.
Trabecular bone width: is used for describing the structural morphology of the trabecular bone and explaining the change of bone mass. The change can affect the bone mass, and under the condition of a certain amount, the wider the width is, the more the bone mass is.
Number of trabeculae: is used for describing the structural morphology of the trabecular bone and explaining the change of bone mass. The change can affect the bone mass, and under the condition of a certain width, the more the bone mass is, the more the number is.
Trabecular bone separation degree: the mean distance between the trabeculae is used to describe the trabecular structure morphology. The greater the separation, the greater the distance between trabeculae and the looser the bone.
Number of trabecular bone connecting points: the number of the cross connection points of the trabecular bone formed into the net shape in a unit area is used for describing the structural form of the trabecular bone and reflecting the connectivity of the trabecular bone net structure.
Number of trabecular free ends: the number of the broken ends of the trabecular bone in the unit visual field is used for describing the structural morphology of the trabecular bone and reflecting the connectivity of the reticular structure of the trabecular bone.
TABLE 2 calculation formula of trabecular bone parameters
Figure BDA0002300918730000091
2.3.3Micro-CT bone microstructure detection: after bone density detection, quickly dissecting and separating bilateral thighbones, shinbones, humerus and 5 th lumbar vertebrae of a rat, removing soft tissues such as muscles and fascias, only keeping bone tissues, wiping the bone tissues cleanly by using sterile gauze, putting the treated thighbones and 5 th lumbar vertebrae of the rat into a small animal Micro-CT instrument, vertically fixing the instrument in a sample fixer along a long axis, setting voltage 60KV and power 40W, starting scanning after correction, scanning the same sample to obtain pictures of different sections, performing three-dimensional reconstruction on the areas (regions of interest, ROI) of the lumbar vertebrae and the thighbones, and performing quantitative analysis by using MicroView software, wherein main analysis parameters comprise trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular distance (Tb.Sp), bone volume fraction (bone volume/volume, BV/TV) and the like, Structure Model Index (SMI).
3. Results of the experiment
3.1 general case
During the observation period of 24 weeks, the rats in each group have white and glossy hair, lively spirit, normal activity and normal diet water inflow, the bone density is detected at 12 weeks, the molding success rate is 100%, 1 rat in each of the model group and the low-dose group dies, and the death rate is 0.03%.
3.2 Effect on bone structural morphology
The direct observation of the optical lens and the micro CT image shows that compared with the false operation group, the bone slice display model group has the advantages that the number of trabeculae is reduced, the trabeculae wall is thinned and thinned, the number of trabeculae connecting points is reduced, the number of free tail ends is increased, the trabecular gap is increased, and the bone marrow cavity is widened, thereby indicating that the replication of the osteoporosis model is successful. Compared with the model group, each group of the formula and the CallQi D control group have the advantages that the trabecular bone structure is obviously improved, namely the number of trabecular bone is increased, the trabecular bone wall is thickened, the number of trabecular bone connecting points is increased, the number of free tail ends is reduced, the trabecular bone gap is reduced, the medullary cavity of the trabecular bone is reduced, and the like (shown in attached figures 1-3).
3.2 Effect on Total body bone Density in groups of rats
The results showed (see table 3) that the model group had significantly lower total body bone density than the sham group, with statistically significant differences (P < 0.01); compared with the model group, the active ingredient composition groups of the traditional Chinese medicine and the calqi group D are obviously higher than the model group, and the difference has statistical significance (P is less than 0.01 or 0.05); compared with the calqi group D, the dosage group and the high dosage group in the traditional Chinese medicine active ingredient formula are both obviously higher than the calqi group D, and have statistically significant difference (P < 0.01).
TABLE 3 comparison of bone Density changes in rats in groups 24 weeks post-surgery
Figure BDA0002300918730000101
Figure BDA0002300918730000102
Note: comparing with model group by adopting one-way variance analysis, wherein # P is less than 0.05; # P <0.01, compared to model group; Δ P <0.01, compared to sham group; & P <0.01, compared to CallQi group D.
3.3 Effect on the variation of the Width of the trabecular bone in rats of various groups
The results show (see table 4) that the trabecular width of the model group is significantly lower than that of the sham group, with statistically significant differences (P < 0.01); compared with the model group, the active ingredient composition groups of the traditional Chinese medicine and the calqi group D are obviously higher than the model group, and the difference has statistical significance (P is less than 0.01 or 0.05); compared with the calqi group D, the dosage group and the high dosage group in the traditional Chinese medicine active ingredient formula are both obviously higher than the calqi group D, and have statistically significant difference (P < 0.01).
TABLE 4 comparison of trabecular Width Change for each group
Figure BDA0002300918730000103
Figure BDA0002300918730000104
Note: comparing with model group by adopting one-way variance analysis, wherein # P is less than 0.05; # P <0.01, compared to model group; Δ P <0.01, compared to sham group; & P <0.01, compared to CallQi group D.
3.4 Effect on the variation of the number of trabeculae in rats in each group
The results show (see table 5) that the trabecular bone number in the model group is significantly lower than in the sham group, with statistically significant differences (P < 0.01); compared with the model group, the active ingredient composition groups of the traditional Chinese medicine and the calqi group D are obviously higher than the model group, and the difference has statistical significance (P is less than 0.01 or 0.05); compared with the CallQi group D, the traditional Chinese medicine active ingredient formula has obviously higher dosage group and high dosage group than the CallQi group D, and has statistically significant difference (P <0.01)
TABLE 5 comparison of trabecular number variation for each group
Figure BDA0002300918730000111
Figure BDA0002300918730000112
Note: comparing with model group by adopting one-way variance analysis, wherein # P is less than 0.05; # P <0.01, compared to model group; Δ P <0.01, compared to sham group; & P <0.01, compared to CallQi group D.
3.5 Effect on the Change in the degree of separation of the trabeculae on rats in Each group
The results show (see table 6) that the trabecular bone separation was significantly higher in the model group than in the sham-operated group with statistically significant differences (P < 0.01); compared with the model group, the active ingredient composition groups of the traditional Chinese medicine and the calqi group D are obviously lower than the model group, and the difference has statistical significance (P is less than 0.01 or 0.05); compared with the CallQi group D, the traditional Chinese medicine active ingredient formula has obviously lower dosage group and high dosage group than the CallQi group D, and has statistically significant difference (P <0.01)
TABLE 6 comparison of trabecular bone separation Change for each group
Figure BDA0002300918730000113
Figure BDA0002300918730000114
Note: comparing with model group by adopting one-way variance analysis, wherein # P is less than 0.05; # P <0.01, compared to model group; Δ P <0.01, compared to sham group; & P <0.01, compared to CallQi group D.
3.6 Effect on the number of points of trabecular bone connection of rats in each group
The results show (see table 7) that the number of trabecular bone junctions in the model group is significantly lower than that in the sham operation group, with statistically significant differences (P < 0.01); compared with the model group, the active ingredient composition groups of the traditional Chinese medicine and the calqi group D are obviously higher than the model group, and the difference has statistical significance (P is less than 0.01 or 0.05); compared with the calqi group D, the dosage group and the high dosage group in the traditional Chinese medicine active ingredient formula are both obviously higher than the calqi group D, and have statistically significant difference (P < 0.01).
TABLE 7 comparison of the number of trabecular bone connections in each group
Figure BDA0002300918730000121
Figure BDA0002300918730000122
Note: comparing with model group by adopting one-way variance analysis, wherein # P is less than 0.05; # P <0.01, compared to model group; Δ P <0.01, compared to sham group; & P <0.01, compared to CallQi group D.
3.7 Effect on the number of free Ends of trabecular bone in rats in Each group
The results show (see table 8) that the number of trabecular free ends in the model group is significantly higher than that in the sham group, with statistically significant differences (P < 0.01); compared with the model group, the active ingredient composition groups of the traditional Chinese medicine and the calqi group D are obviously lower than the model group, and the difference has statistical significance (P is less than 0.01 or 0.05); compared with the calqi group D, the dosage group and the high dosage group in the traditional Chinese medicine active ingredient formula are both obviously lower than the calqi group D, and have statistically significant difference (P < 0.01).
TABLE 8 comparison of changes in the number of trabecular free ends in each group
Figure BDA0002300918730000123
Figure BDA0002300918730000124
Note: comparing with model group by adopting one-way variance analysis, wherein # P is less than 0.05; # P <0.01, compared to model group; Δ P <0.01, compared to sham group; & P <0.01, compared to CallQi group D.
4. Conclusion
The traditional Chinese medicine active ingredient formula can improve the bone density of postmenopausal osteoporosis model rats to different degrees by using various dosage groups and calcium and vitamin D, so that the number of trabeculae is increased, the trabecular wall is thickened, the number of trabecular connection points is increased, the number of free terminal ends is reduced, the gap between the trabeculae is reduced, the medullary cavity of the trabeculae is reduced, and the like, and particularly the high dosage group of the traditional Chinese medicine active ingredient formula is remarkable.
Effect example 2 study of adverse drug reactions
In the acute toxicity test of the embodiment of the combination formula, the formula of the group 1 is adopted, the administration dosage is determined according to the acute toxicity pre-test (see table 9), the drug suspension is prepared, the animals adopt SPF grade SD rats 60, the age of 3 months, the weight of male mice is 350 +/-30 g, the weight of female mice is 250 +/-30 g (animal qualification number: SCXK (Guangdong) 2013 and 0002, the center of Guangdong province experimental animals), the groups are randomly divided into 6 groups, each group is 10, and the male and female halves are respectively a blank control group and a Chinese medicine active ingredient formula 1, 2, 3, 4 and 5 groups. Before the experiment, after fasting and water prohibition are carried out for 12 hours, the dosage group is 10 mL/kg -1 The weight is administrated by gavage for 1 time, the dosage within 1 day is calculated, before use, distilled water is used for preparing suspension, and the control group is administrated with distilled water with the same volume as the gavage for acute toxicity test observation.
After the administration of the medicines by intragastric administration, 5 normal rearing are carried out in each cage, and the observation is carried out for 1 time every 15min within 2h after the administration; observing for 1 time every 30min within 2-4 h after administration; observing for 1 time every 1h within 4-8 h after administration; observing for 1 time every 4 hours within 8-24 hours after administration; after the administration, the rats are observed once a day from the 2 nd day, the weight is weighed, and the motility, the drinking condition, the motility, the abnormal muscle movement, the pupil change, the eyeball bulge, the eyelid droop, the external reaction, the abnormal secretion, the abnormal stool and urine, the abnormal breathing, the skin color change and other toxic reactions and death conditions which may occur in 14d of each rat are closely observed. If the dead rat is dead, the dead rat is dissected, the change of organs such as heart, liver, kidney, lung, brain, spleen, stomach, small intestine and the like is observed by naked eyes, after the 14d observation period is finished, the living rat in each group is sacrificed and dissected, and the gross pathological changes of main organs are observed by naked eyes according to the same method.
The cumulative death number of rats in different time and the cumulative death-time condition of rats after different doses of the traditional Chinese medicine active ingredient formula for intragastric administration to rats are shown in table 9. Calculating traditional Chinese medicine active ingredient formula LD according to death number and administration dose of rats in each dose group 50 And 95% confidence limits, see table 10.
TABLE 9 cumulative mortality of rats at different times after gavage with Chinese herbal active ingredient
Figure BDA0002300918730000131
Figure BDA0002300918730000141
TABLE 10 administration of active ingredient formula of Chinese medicine by intragastric administration LD 50 And 95% confidence limit
Figure BDA0002300918730000142
The death of rats caused by the traditional Chinese medicine active ingredient formula mostly occurs 1-3 days, and no death phenomenon is found in the surviving animals after 4 days. Maximum administration dose LD for intragastric administration of Chinese medicinal active ingredient formula 100 4500.0mg kg -1 ·d -1 Minimum dose LD 0 2644.8 mg/kg -1 ·d -1 The calculated half lethal dose LD is processed by adopting a Bliss calculation method 50 A value of 3256.6mg kg -1 ·d -1 The 95% confidence limit is 3056.4-3457.9 mg/kg -1 ·d -1 。
Observation of acute toxicity symptoms and body weight change in rats: after the administration, the activity of the rats is reduced and quiet, and as the time increases, individual rats are unstable in walking, lie on the back, are lethargic, are congested in limbs and tails and die gradually due to intermittent convulsion, the death of the rats mostly occurs in 1-3 days, the symptoms are relieved after the survival of the rats lasts for more than 4 days, and the rats gradually return to normal after 4-7 days. Activities, food intake, drinking and the like are basically recovered to be normal after 7-8 days of administration. Normal rats are dissected, and the changes of the major organs such as heart, liver, spleen, lung, kidney, liver and the like are observed under naked eyes, and no obvious abnormality is found. The surviving rats after gavage had no significant change in body weight during the observation period.
In pharmacodynamic experiments, the active ingredients of the traditional Chinese medicine are adopted to prepare low, medium and high dose groups (the formula of the embodiment 1) 36.0, 72.0 and 114.0mg/kg -1 ·d -1 And (3) performing intragastric administration, preparing a suspension by using normal saline before use, continuously performing intragastric administration for 12 weeks for 1 time every day, and ensuring that the experimental rat has no obvious abnormality in urination, defecation, drinking water, food intake, blood routine, urine routine and liver and kidney functions.
In conclusion, the traditional Chinese medicine active ingredient formula disclosed by the invention has a better curative effect on postmenopausal osteoporosis, can improve the bone mineral density, improve the bone microstructure and the like, is particularly remarkable in high dosage, has the advantages of clear ingredients, small dosage form, convenience in carrying and the like, is an innovation in medicine for treating postmenopausal osteoporosis, and has a higher practical value.
The above-mentioned embodiments are only preferred embodiments of the present invention, and not intended to limit the present invention in any way, and although the present invention has been disclosed by the above-mentioned preferred embodiments, the present invention is not limited thereto, and those skilled in the art can make modifications or changes to equivalent embodiments by using the technical contents disclosed above without departing from the scope of the present invention, but all simple modifications, equivalents and changes made to the above-mentioned embodiments according to the technical essence of the present invention are within the scope of the present invention.

Claims (8)

1. A traditional Chinese medicine active ingredient prescription for treating postmenopausal osteoporosis is characterized in that,
the formula consists of the following components in percentage by weight:
wolfberry polysaccharide: icariin: polydatin: and (3) quercetin: sodium danshensu =50:6:50:1: 10.
2. The active ingredient formulation of a traditional Chinese medicine for treating postmenopausal osteoporosis of claim 1, wherein:
the lycium barbarum polysaccharide is determined to have a purity of more than 90% by high performance liquid chromatography.
3. The active ingredient formulation of a traditional Chinese medicine for treating postmenopausal osteoporosis of claim 1, wherein:
the icariin, polydatin, quercetin or salvianic acid A sodium are icariin, polydatin, quercetin or salvianic acid A sodium with content not lower than 98% determined by high performance liquid chromatography.
4. The use of the traditional Chinese medicine active ingredient composition for the treatment of postmenopausal osteoporosis of claim 1 in the preparation of a medicament for the treatment of postmenopausal osteoporosis.
5. The use of the traditional Chinese medicine active ingredient composition for the treatment of postmenopausal osteoporosis according to claim 4, wherein the traditional Chinese medicine active ingredient composition is used for preparing a medicine for the treatment of postmenopausal osteoporosis, and is characterized in that:
the medicine for treating postmenopausal osteoporosis contains one or more pharmaceutically acceptable auxiliary materials.
6. The use of the traditional Chinese medicine active ingredient composition for the treatment of postmenopausal osteoporosis of claim 5 in the preparation of a medicament for the treatment of postmenopausal osteoporosis, wherein:
the adjuvant is at least one of sustained release agent, binder, humectant, disintegrating agent, absorption enhancer, surfactant, antibacterial agent, aromatic agent, antioxidant, pH regulator, protectant, diluent, lubricant, solvent, matrix or carrier material.
7. The application of the traditional Chinese medicine active ingredient composition for treating postmenopausal osteoporosis according to any one of claims 4 to 6 in the preparation of a medicine for treating postmenopausal osteoporosis is characterized in that:
the dosage form of the medicine for treating postmenopausal osteoporosis is an oral preparation.
8. The use of the traditional Chinese medicine active ingredient composition for the treatment of postmenopausal osteoporosis according to claim 7, wherein the traditional Chinese medicine active ingredient composition is used for preparing a medicine for the treatment of postmenopausal osteoporosis, and is characterized in that:
the oral preparation is granules, tablets, pills, capsules or oral liquid.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1480142A (en) * 2003-06-30 2004-03-10 广东医学院 compound preparation composed of longspur epimedium and estrogen for treating osteoporosis of women after climacteric
CN1539427A (en) * 2003-04-23 2004-10-27 山东绿叶天然药物研究开发有限公司 Icariin in application of preparing medication for treating and preventing osteoporosis
CN108042795A (en) * 2017-12-29 2018-05-18 哈尔滨工业大学 A kind of Radix Salviae Miltiorrhizae bone invigorating composition

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1539427A (en) * 2003-04-23 2004-10-27 山东绿叶天然药物研究开发有限公司 Icariin in application of preparing medication for treating and preventing osteoporosis
CN1480142A (en) * 2003-06-30 2004-03-10 广东医学院 compound preparation composed of longspur epimedium and estrogen for treating osteoporosis of women after climacteric
CN108042795A (en) * 2017-12-29 2018-05-18 哈尔滨工业大学 A kind of Radix Salviae Miltiorrhizae bone invigorating composition

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
植物雌激素类单味中药抗绝经后骨质疏松实验研究进展;赵军等;《云南中医中药杂志》;20191020;第40卷(第10期);参见第1.3节 *
槲皮素通过抗骨相关细胞衰老作用治疗雌激素缺乏骨质疏松症的初步研究;顾艺婧等;《同济大学学报（医学版）》;20190625;第40卷(第3期);参见摘要 *
绝经后骨质疏松症的中医常用治法及四味中药的研究进展;魏戌等;《环球中医药》;20111106;第4卷(第6期);参见2.1节，2.3节和2.4节 *

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