CN1416873A - A pharmaceutical composition for treating osteoporosis, and its preparation method - Google Patents
A pharmaceutical composition for treating osteoporosis, and its preparation method Download PDFInfo
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- CN1416873A CN1416873A CN02149471A CN02149471A CN1416873A CN 1416873 A CN1416873 A CN 1416873A CN 02149471 A CN02149471 A CN 02149471A CN 02149471 A CN02149471 A CN 02149471A CN 1416873 A CN1416873 A CN 1416873A
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- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Medicinal Preparation (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a pharmaceutical composition for treating osteoporosis and a preparation method thereof, wherein the pharmaceutical composition is prepared from epimedium, oyster or extracts thereof. The preparation method comprises the steps of taking epimedium leaves, boiling the epimedium leaves with water, precipitating the epimedium leaves with ethanol, and refining filtrate by a polyamide adsorption method to obtain an epimedium extract; hydrolyzing Concha Ostreae acid to obtain Concha Ostreae hydrolysate; mixing the above two parts to obtain the pharmaceutical composition. The pharmaceutical composition has definite curative effect on osteoporosis.
Description
Technical field
The present invention relates to a kind of pharmaceutical composition and preparation method thereof, particularly relate to a kind of medicine for the treatment of osteoporosis and preparation method thereof.
Background technology
Osteoporosis is that a kind of bone amount minimizing and osseous tissue microstructure are impaired, causes the systemic skeletal diseases that skeleton fragility increases and the risk of fractures degree increases then, often shows as pain, height shortening, hunchback, fracture etc. clinically.
Summary of the invention
Pharmaceutical composition of the treatment osteoporosis that the object of the invention is to provide a kind of process stabilizing, quality controllable, determined curative effect, have no side effect and preparation method thereof.
The present invention seeks to be achieved through the following technical solutions:
Herba Epimedii: Concha Ostreae 15-25: 1-1.5, preferred proportion are Herba Epimedii: Concha Ostreae 20: 1; Get Folium Epimedii and decoct with water 2-4 time, each 1-2 hour, add 10-20 times of water gaging at every turn, decocting liquid merges, and filters, and filtrate is concentrated into the clear paste that relative density is 1.02-1.04 (60 ℃), add ethanol and make the alcohol amount of containing be 60-80%, left standstill 12-48 hour, get supernatant and reclaim ethanol to there not being the alcohol flavor, add water to medicinal liquid: medical material is 2: 1, adds hydrochloric acid to pH=2-5, cold preservation, placed 24-72 hour, filter, precipitation is dry, and is standby; Filtrate is refining with the polyamide absorption method, polyamide column absorption method condition: blade diameter length ratio is 1: 2-5, liquor strength are by medical material: medicinal liquid is 1: 1-3, pH=2-5, than applied sample amount is medical material: polyamide 2-5: 1, wash 3-6 times of column volume, the 60-80% ethanol elution is to the eluent color burn, begin to collect eluent, collect 1-3 times of column volume, reclaim ethanol, concentrated, dry; Wash 5-10 times of column volume, behind the eccysis ethanol, repeat sample; Polyamide renovation process: repeat 2-5 back regeneration of sample 1 time, behind 2-5 times of column volume eccysis absorption of 0.5-1% sodium hydrate aqueous solution impurity, wash, to effluent pH=2-4, repeat sample with the pH=2-4 aqueous hydrochloric acid solution; Renewable 2 times of polyamide is gone up sample 9 times altogether; The per kilogram polyamide can be made with extra care Herba Epimedii 15-30 kilogram; The 60-80% ethanol elution, eluent reclaims ethanol, and being concentrated into relative density is 60 ℃ of thick pastes of 1.20～1.25 down, drying; The dried cream of two parts is merged, pulverize, get Herba Epimedii extract, standby; Get Concha Ostreae, 60 mesh sieves are crossed in pulverizing, add the acetic acid that 8-12 doubly measures 20-30%, and heating hydrolysis 3-6 hour, filter, filtrate decompression is concentrated into semisolid, and drying is pulverized, and it is standby to get the Concha Ostreae hydrolysate.Above-mentioned two parts are mixed, pharmaceutical composition, can be directly or add excipient and make clinical acceptable forms with pharmaceutical composition, as tablet, capsule, oral liquid, granule, effervescent, buccal tablet etc.
Pharmaceutical composition of the present invention also can be directly surpasses 50% Herba Epimedii total flavones or contains the Herba Epimedii effective part extract that surpasses 15% icariin by containing: contain the Concha Ostreae effective site hydrolysate 1-2 that surpasses 75% calcium acetate: 1-3 forms; Herba Epimedii total flavones mainly contains icariin, epimidin C, epimidin B, arrow icariin B, precious icariin I, and effective ingredient such as precious icariin II, Flos Caryophylli icariin C, Flos Caryophylli icariin F, icariside I wherein contain and surpass 15% icariin.Preferred proportion is 1: 2.
The method of quality control of pharmaceutical composition of the present invention is characterized in that this method is:
Differentiate: get aforementioned pharmaceutical compositions 0.2g, add each 1ml of sulphuric acid and ethanol, heat, the fragrance of ethyl acetate promptly takes place; Get broken mistake 80 mesh sieves of aforementioned pharmaceutical compositions, take by weighing 0.05g, add ethanol 10ml, ultrasonic 4-8 minute, filter, filtrate evaporate to dryness, residue add ethanol 1-2ml makes dissolving, as need testing solution; Other gets the icariin reference substance, adds methanol and makes the solution that every 1ml contains 0.1mg, in contrast product solution; According to thin layer chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, with 8-11: 0.8-1.2: 0.8-1.2: 0.8-1.2 ethyl acetate-butanone-formic acid-water is developing solvent, launch, take out, dry, spray is with 8-12% sulphuric acid ethanol liquid, in 100-105 ℃ of heating 2-4 minute; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical yellow spotting;
Assay: icariin is according to high effective liquid chromatography for measuring; Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler; 26-30: the 70-74 acetonitrile-water is a mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the icariin peak should be not less than 1500; The reference substance solution preparation: it is an amount of that precision takes by weighing the icariin reference substance, adds the reference substance solution that methanol is made 5 μ g/ml, promptly; The need testing solution preparation: get aforementioned pharmaceutical compositions and pulverized 80 mesh sieves, precision takes by weighing 0.1g, puts in the 100ml tool plug conical flask, the accurate 40-60% ethanol 45-55ml that adds, close plug claims to decide weight, ultrasonic to dissolving, claim again to decide weight, supply the weight that subtracts mistake with 40-60% ethanol, shake up, filter, get subsequent filtrate 0.5ml, put in the 10ml measuring bottle, add methanol to scale, shake up, as need testing solution; Algoscopy: accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing, inject chromatograph of liquid, measure, promptly; Pharmaceutical composition of the present invention contains Herba Epimedii in icariin, must not be less than 50mg/g;
Calcium acetate is got aforementioned pharmaceutical compositions and pulverized 80 mesh sieves, and precision takes by weighing 0.5g and puts in the crucible, and is slowly blazing to carbonization fully, puts and is chilled to room temperature; Make complete ashing at 500-700 ℃ of blazing 3h again, put coldly, residue adds dilute hydrochloric acid 4-8ml makes dissolving, put in the 10ml measuring bottle, thin up shakes up to scale, the accurate 1ml diluent of drawing adds water 100ml, 0.05% methyl red indicator 1ml, drip 10% potassium hydroxide to solution to light yellow, continue to add 10ml again, add calcein 0.1g, with the titration of disodiumedetate volumetric solution, to the yellow-green fluorescence disappearance of solution, and be orange, promptly; Every 1ml disodiumedetate volumetric solution is equivalent to the calcium acetate (Ca (Ac) of 15.8mg
2); Pharmaceutical composition of the present invention contains Concha Ostreae must not be less than 0.5g/g in calcium acetate.
Pharmaceutical composition of the present invention (casting capsule) shows that through pharmacodynamic study it is hyperfunction unusually because of estrogen level reduces the bone resorption, the bone formation that cause that this pharmaceutical composition can suppress rat; The bone resorption that suppresses to irritate the stomach retinoic acid and cause is hyperfunction, promotes bone formation; Increase the bone amount; Improve the microstructure of osseous tissue; Improve the biomechanical strength of osseous tissue; Thereby has significant function of resisting osteoporosis.This pharmaceutical composition also has stronger analgesic activity and antiinflammatory and immunological enhancement.
Following experimental example or embodiment are used to further specify the present invention.Experimental example 1, casting capsule are to the influence of castrated rats osteoporosis experimental model
Grouping and administration: get 65 of female sd inbred rats.10 rats of picked at random are sham operated rats (n=10).All the other rats were cooked ovariectomy after 5 days, were divided at random: dosage group (n=11), casting capsule small dose group (n=11) in osteoporosis model group (n=11), positive control drug GUSHUKANG KELI group (n=11), the casting heavy dose of group of capsule (n=11), the casting capsule.It is 1.33% beta-schardinger dextrin-aqueous solution that sham operated rats, model group rat give concentration every day, positive drug group rat gives GUSHUKANG KELI 2.7g/kg (for 8 times of the clinical consumption per day of adult) every day, and the large, medium and small dosage group of casting capsule rat is cast capsule dosage every day respectively and is respectively 400mg/kg, 200mg/kg, 100mg/kg (be respectively the clinical consumption per day of adult 8,4,2 times).Gastric infusion is 3 months continuously.
Modeling method: each organizes rat with 10% chloral hydrate 350mg/kg intraperitoneal injection of anesthesia, cuts the otch of 1.5cm size over against the position of ovary in the spinal column both sides, separates muscle of back, exposes posterior peritoneum, takes out bilateral ovaries after cutting off peritoneum.Sham operated rats is left intact and puts back to the abdominal cavity after ovary is taken out, and sews up wound, sterilization; The operation group is excised after with the bilateral ovaries ligation, sews up wound, sterilization.Sham operated rats is given normal feedstuff, gives low calcium feedstuff, freely drinks water for all the other five groups.Raise after 3 months and put to death.
Observation index: administration finishes, and each treated animal is anaesthetized through 10% chloral hydrate 350mg/kg, draws materials.
(1) body weight: take by weighing body weight weekly a time.
(2) organ index: rat is won its heart, lung, liver,spleen,kidney, uterus after putting to death; The AND scales/electronic balance weighing.The organ index computing formula is as follows:
(3) blood calcium, serium inorganic phosphorus and blood alkali phosphatase:
The common carotid artery intubate is got blood, preserved 4 hours for 4 ℃, 3000 rev/mins centrifugal 10 minutes, separation of serum.The OCPC method is adopted in blood calcium determination, and serium inorganic phosphorus is measured the molybdic acid method that adopts, and King's method is adopted in serum alkaline phosphatase determination, all by the procedure operation of test kit description, measures with the MD-100 automatic biochemical analyzer.Serum calcium, phosphorus, alkaline phosphatase enzyme reagent kit provide by Beijing Zhongsheng Biological Engineering High Technology Company.
(4) urinary hydroxyproline (Hydroxyproline, HOP), creatinine
Rat is put to death and goes into metabolic cage the previous day, fasting, and normal drinking-water is collected its twenty-four-hour urine liquid (24 ± 2 ℃ of temperature, relative humidity 50%).(Hydroxyproline, mensuration HOP) adopts improvement chloramine-T oxidizing process to measure to urinary hydroxyproline
[2]The hydroxyproline standard substance, biochemical institute provides by Chinese Academy of Sciences Shanghai.Picric acid method is adopted in creatinine assay, presses the procedure operation of test kit description, measures with the MD-100 automatic biochemistry analyzer.The creatinine reagent box is provided by Beijing Zhongsheng Biological Engineering High Technology Company.
(5) serum hormone (estradiol, calcitonin, Bone Gla protein):
The common carotid artery intubate is got blood, preserved 4 hours for 4 ℃, 3000 rev/mins centrifugal 10 minutes, separation of serum.Estradiol uses estradiol radioimmunological kit (Depew, Tianjin biotechnology and medical product company limited provide), measures and presses the operation of test kit description; Calcitonin uses calcitonin to put to exempt from medicine box (Science and Technology Development Center of Chinese People's Liberation Army General Hospital put exempt from Research Institute, provide), measures press the medicine box description and operate; Bone Gla protein uses Bone Gla protein to put to exempt from medicine box (Science and Technology Development Center of Chinese People's Liberation Army General Hospital put exempt from Research Institute, provide), measures press the medicine box description and operate.
(6) bone density:
After rat is put to death, strip its left side femur, pick os purum muscle and soft tissue on every side, use the normal saline scrub, wrap up respectively with the normal saline gauze, bone density and biomechanics of bone intensity are surveyed in-40 ℃ of preservations fully.NORLAND X2-36 type dual-energy x-ray borne densitometers is surveyed femoral bmd (BoneMineral Density, BMD, g/cm
2).The left side femur faced upward place the enterprising line scanning of poly (methyl methacrylate) plate, step pitch 0.5 * 0.5mm; Scanning speed 30mm/s; Sweep length 6.55cm.After bone densitometry finishes, will femur-40 ℃ preservation, send and survey biomechanics of bone intensity.
(7) biomechanics of bone intensity:
Place the WD-1 type electronic universal tester to make the three-point bending mechanical test femur, span 25mm, loading velocity 2mm/min, record load-deflection curve.Calculate maximum load, maximum oar degree, bending rigidity, energy absorption from curve.
(8) bone inorganic element content:
Rat strips its right side femur after putting to death, and picks os purum muscle and soft tissue on every side, uses the normal saline scrub.Sample is delivered to test center of Beijing Normal University, produces the content of inorganic elementss such as calcium, phosphorus, magnesium, zinc, aluminum in the Jarrell-Ash ICAP9000 type inductively coupled plasma spectrophotometer bone with the U.S..
(9) vertebra osseous tissue morphometry:
Rat was put to death preceding the 14th day, irritated stomach tetracycline (Tetracycline Amersham is available from Huamei Bio-Engrg Co.) 35～40mg/kg body weight, continuous two days.After being separated by 10 days, two days tetracyclines of continuous irrigation stomach in kind again.After the execution, get third and fourth lumbar vertebra, after fixing, dehydration, embedding etc. are handled,, prepare vertical undecalcified bone slice of 5 μ m, Toluidine blue staining with LEICA 2163 type undecalcified bone slice machines.Toluidine blue staining is not made in the section that is used for Fluirescence observation.With Olympus BH-2 microscope, the observation of JVCPK-C1380 image pick-up card adopts bone histomorphometry's analysis software (Chinese People's Liberation Army General Hospital's orthopaedics Research Institute) that observation index is measured and analyzes.The parameters that adopts is: bone trabecula surface percentage, bone formation surface percentage, bone resorption surface percentage, average bone wall thickness and or mineralising deposition.
(10) the bone formation morphosis is observed
Strip the left side femur, pick os purum muscle and soft tissue on every side, use the normal saline scrub; Fix more than 24 hours with 4% formalin for 4 ℃, to appropriate level, get on the proximal femur hip joint head maximum area coronal section stringer and cut routine paraffin wax embedding, section, HE dyeing, Olympus microscopic examination, microphotograph open with the decalcification of EDTA decalcifying Fluid.
Result of the test: all (X ± SD) expression relatively adopts SAS statistical software generalized linear model to carry out covariance analysis to result of the test between group, have the standard of significance as difference with p＜0.05 with mean+SD.N is the rat number.In the experimentation, because traumatic infection causes indivedual rats deaths.It is different when so the number of rat is with the test grouping in the result of the test.
1, body weight change
Table 1 the weight of animals change list
(N) group after 14 weeks of (N) administration before the dosage administration
(g/kg) (g (only)) (g (only)) sham operated rats-282.0 ± 14.6 (10) 366.0 ± 29.5 (10)
* * △ △ △Model group-286.4 ± 16.6 (11) 440.0 ± 54.8 (10)
* * ZeroGUSHUKANG KELI group 2.7 280.5 ± 12.1 (11) 394.4 ± 55.4 (9)
* * △Casting capsule group 0.4 285.0 ± 13.4 (11) 387.5 ± 19.1 (8)
* * △Casting capsule group 0.2 288.6 ± 15.0 (11) 411.9 ± 50.4 (8)
* *Casting capsule group 0.1 286.8 ± 18.1 (11) 420.0 ± 28.6 (9)
* *
Annotate: 1. compare p＞0.05 between each group before the administration.
2. compare after the administration with before the administration,
* *P＜0.001.
3. compare with model group after the administration,
△ △ △P＜0.001,
△P＜0.05; Compare with the heavy dose group,
○p＜0.05。
Last table shows: each organizes the preceding body weight there was no significant difference of rat administration, and comparability is arranged between group.After the rat spay, because estrogen level descends rapidly, cause the rapid accumulation of subcutaneous fat, therefore, body weight increases rapidly.After casting the capsule heavy dose, the trend that rat body weight increases rapidly is controlled, shows that the casting capsule can suppress the accumulation of subcutaneous fat after the ovariectomized rats to a certain extent.
2, respectively organize the exponential comparison of Rats Organs and Tissues
Table 2 is respectively organized Rats Organs and Tissues index (g/100g)
Agent group N cardiopulmonary liver spleen kidney uterus
Amount sham operated rats-10 0.25 ± 0.04 0.30 ± 0.03 2.28 ± 0.47 0.14 ± 0.02 0.45 ± 0.07 0.069 ± 0.013
* * △ △ △Model group-10 0.24 ± 0.03 0.29 ± 0.04 2.11 ± 0.26 0.14 ± 0.03 0.44 ± 0.05 0.018 ± 0.005
△ 000GUSHUKANG KELI group 2.7 9 0.24 ± 0.03 0.27 ± 0.04 2.11 ± 0.36 0.15 ± 0.04 0.44 ± 0.03 0.022 ± 0.004
000Casting capsule group 0.4 8 0.25 ± 0.05 0.28 ± 0.06 2.16 ± 0.36 0.15 ± 0.02 0.46 ± 0.06 0.025 ± 0.008
* 000Casting capsule group 0.2 8 0.23 ± 0.04 0.28 ± 0.05 2.23 ± 0.44 0.14 ± 0.03 0.43 ± 0.05 0.021 ± 0.006
000Casting capsule group 0.1 9 0.24 ± 0.03 0.30 ± 0.04 2.14 ± 0.33 0.16 ± 0.03 0.47 ± 0.05 0.019 ± 0.005
000
Annotate: 1. compare with model group,
* *P＜0.001;
*P＜0.05.
2. compare with the heavy dose group,
△ △ △P＜0.001;
△P＜0.05.
3. compare with the blank group:
000P＜0.001.
Remove ovary and can cause the rat uterus atrophy, the uterus index obviously reduces.Relatively cast each administration group of capsule with model group and can improve the uterus index of removal ovary rat in various degree; Heavy dose of group has significant difference.
3, serum calcium, phosphorus, alkali phosphatase and urine proline/creatinine table 3 medicine are to the influence of rat model serum calcium, phosphorus, alkali phosphatase and urine proline/creatinine
Dosage N Ca P ALP group HOP/Cr
(g/kg) (only) (mmol/L) (mmol/L) (U/L) sham operated rats-10 2.70 ± 0.35 1.38 ± 0.19 49.0 ± 5.6
* *0.92 ± 0.11
* *Model group-10 2.53 ± 0.35 1.40 ± 0.16 68.7 ± 5.8
△ △ △2.34 ± 0.30
△ △ △GUSHUKANG KELI group 2.7 9 2.70 ± 0.33 1.44 ± 0.18 57.9 ± 6.0
* *1.27 ± 0.12
* * △ △Casting capsule group 0.4 8 2.74 ± 0.26 1.40 ± 0.19 53.3 ± 6.8
* *1.00 ± 0.13
* *Casting capsule group 0.2 8 2.54 ± 0.25 1.40 ± 0.13 60.6 ± 7.6
* △1.78 ± 0.25
* * △ △ △Casting capsule group 0.1 9 2.64 ± 0.26 1.43 ± 0.18 66.8 ± 4.8
△ △ △1.94 ± 0.20
* * △ △ △
Annotate: 1. compare with model group,
* *P＜0.001;
*P＜0.01.
2. compare with the heavy dose group,
△ △ △P＜0.001;
△ △P＜0.01;
△P＜0.05.
The Serum alkaline phosphatase activity of model group rat (ALP) all raises than blank group with urinary hydroxyproline/creatinine (HOP/Cr), and significant difference is arranged.Show that castration causes that bone formation and bone resorption strengthen simultaneously, but bone resorption is relatively greater than bone formation, the bone amount can not be kept balance and cause osteoporosis rat.
Casting big or middle dosage group of capsule and GUSHUKANG can make alkaline phosphatase activities reduce than model group; Casting large, medium and small dosage group of capsule and GUSHUKANG all can make urinary hydroxyproline/creatinine reduce than model group; Significant difference is all arranged.The effect of the heavy dose of group of casting capsule is stronger than other treatment group, and significant difference is arranged.
Prompting casting capsule can improve the alkali base acid phosphatase of castration osteoporosis model rat and the supernormal growth of urinary hydroxyproline/creatinine.
4, serum hormone
Table 4 medicine is to the influence of rat hormonal readiness
Dosage N estradiol calcitonin Bone Gla protein group
(g/kg) (only) (ng/L) (ng/L) (μ g/L) sham operated rats-10 14.11 ± 1.46
* * △ △238.4 ± 19.3
* * △ △ △1.45 ± 0.17
* * △ △ △Model group-10 7.01 ± 0.99
△ △ △156.8 ± 22.7
△ △0.82 ± 0.09
△ △ △GUSHUKANG KELI group 2.7 9 11.52 ± 1.65
* *184.2 ± 18.2
* △1.05 ± 0.13
* * △ △Casting capsule group 0.4 8 12.42 ± 1.23
* *205.5 ± 21.6
* *1.23 ± 0.10
* *Casting capsule group 0.2 8 8.40 ± 0.87
* △ △ △173.4 ± 16.9
△ △0.89 ± 0.11
△ △ △Casting capsule group 0.1 9 7.73 ± 1.01
△ △ △167.8 ± 18.4
△ △ △0.84 ± 0.05
△ △ △
Annotate: 1. compare with model group,
* *P＜0.001;
*P＜0.01;
*P＜0.05.
2. compare with the heavy dose group,
△ △ △P＜0.001;
△ △P＜0.01;
△P＜0.05.
Above-mentioned three hormone serum levels of model group rat reduce than blank group, and significant difference is arranged.
Casting big or middle dosage group of capsule and GUSHUKANG group can make serum estradiol content raise than model group; Heavy dose of group of casting capsule and GUSHUKANG group can make serum calcitonin, BGP content raise than model group; Significant difference is all arranged.The heavy dose of group of casting capsule to the therapeutical effect of estradiol be better than wherein, small dose group, the heavy dose of group of casting capsule is better than other treatment group to the improvement effect of serum calcitonin, Bone Gla protein.This results suggest casting capsule and GUSHUKANG all can improve the hormone serum level of osteoporosis model rat.
5, femoral bmd
Table 5 medicine influences group dosage (g/kg) N (only) bone density (g/cm to the rat femur bone density
2) sham operated rats-10 0.1597 ± 0.0033
* * △Model group-10 0.1443 ± 0.0068
△ △GUSHUKANG KELI group 2.7 9 0.1525 ± 0.0052
*Casting capsule group 0.4 8 0.1536 ± 0.0065
*Casting capsule group 0.2 8 0.1 494 ± 0.0056 casting capsule group 0.1 9 0.1480 ± 0.0064
△
Annotate: 1. compare with model group,
* *P＜0.001;
*P＜0.01.
2. compare with the heavy dose group,
△ △P＜0.01;
△P＜0.05.
The rat castration can cause that bone density obviously reduces, and forms osteoporosis, shows the model success.
After casting capsule heavy dose and GUSHUKANG treatment, all the bone density than model group rat significantly raises.
This results suggest casting capsule and GUSHUKANG all can increase the bone density of castration osteoporosis model rat.
6, femur inorganic elements
Table 6 medicine is to the influence of rat femur inorganic element content
Dosage N Ca P Zn Mg group
(g/kg) (only) (mg/g) (mg/g) sham operated rats-10 198.2 ± 7.9 of (mg/g) (μ g/g)
* *64.1 ± 2.3 214.7 ± 5.1
* * △ △3.47 ± 0.27
* *Model group-10 178.7 ± 7.9
△ △ △62.0 ± 3.6 191.2 ± 4.3
△ △ △2.32 ± 0.32
△ △ △GUSHUKANG KELI group 2.7 9 189.0 ± 4.7
* △63.9 ± 4.2 201.6 ± 12.1
*3.04 ± 0.15
* * △Casting capsule group 0.4 8 195.8 ± 3.4
* *63.9 ± 1.4 204.5 ± 6.9
* *3.40 ± 0.28
* *Casting capsule group 0.2 8 187.8 ± 6.8
* △64.3 ± 3.0 199.7 ± 10.0
*2.99 ± 0.32
* * △ △Casting capsule group 0.1 9 184.5 ± 7.8
△ △63.6 ± 2.7 195.7 ± 7.3
△2.81 ± 0.33
* * △ △ △
Annotate: 1. compare with model group,
* *P＜0.001;
*P＜0.01;
*P＜0.05.
2. compare with the heavy dose group,
△ △ △P＜0.001;
△ △P＜0.01;
△P＜0.05.
Osteoporosis rat due to the removal ovary, the content of calcium, magnesium, zinc all significantly reduces in the osseous tissue.
Casting big or middle dosage group of capsule and GUSHUKANG group can obviously improve the content of bone calcium, zinc; Casting large, medium and small dosage group of capsule and GUSHUKANG group can obviously improve the content of bone magnesium; Significant difference is all arranged.The effect that the heavy dose of group of casting capsule improves bone calcium, bone magnesium obviously is better than other treatment group.
Prompting casting capsule has can suppress bone resorption, reduces bone loss; Can promote absorption and the utilization of body to calcium; And multiple inorganic elementss such as magnesium, zinc are provided, to help the effects such as deposition in alcium and phosphor metabolization and bone ore deposit.
7, femur biomechanics
Table 7 medicine is to the influence of rat femur biomechanical strength
The maximum oar degree of dosage N maximum load bending rigidity energy absorption group
(g/kg) (only) (N) (mm) (N/mm) (Nmm) sham operated rats-10 118.1 ± 9.9
* *0.84 ± 0.08
* *180.3 ± 23.8
* *43.5 ± 5.6
* *Model group-10 91.3 ± 8.0
△ △0.63 ± 0.04
△ △ △146.1 ± 12.5
△ △ △25.5 ± 3.1
△ △ △GUSHUKANG KELI group 2.7 9 101.2 ± 8.5
* △0.73 ± 0.07
* △163.2 ± 17.2
*35.3 ± 3.9
* * △ △Casting capsule group 0.4 8 110.6 ± 10.3
* *0.81 ± 0.06
* *175.9 ± 22.0
* *41.5 ± 5.3
* *Casting capsule group 0.2 8 99.0 ± 7.4
△0.71 ± 0.09
* △ △160.0 ± 12.7 32.2 ± 3.8
* △ △ △Casting capsule group 0.1 9 96.9 ± 8.7
△ △0.69 ± 0.08
△ △151.5 ± 14.4
△ △27.6 ± 3.0
△ △ △Casting capsule group 0.4 8 110.6 ± 10.3
* *0.81 ± 0.06
* *175.9 ± 22.0
* *41.5 ± 5.3
* *Casting capsule group 0.2 8 99.0 ± 7.4
△0.71 ± 0.09
* △ △160.0 ± 12.7 32.2 ± 3.8
* △ △ △Casting capsule group 0.1 9 96.9 ± 8.7
△ △0.69 ± 0.08
△ △151.5 ± 14.4
△ △27.6 ± 3.0
△ △ △
Annotate: 1. compare with model group,
* *P＜0.001;
*P＜0.01;
*P＜0.05.
2. compare with the heavy dose group,
△ △ △P＜0.001;
△ △P＜0.01;
△P＜0.05.
Above-mentioned every index of model group rat all significantly is lower than the blank group, and less external force and energy can make fracture take place, and show that removal ovary makes osteoporosis rat, and bone structure changes, flexibility decrease, and fracture risk increases.
Heavy dose of group of casting capsule and GUSHUKANG can improve maximum load, bending rigidity; Casting big or middle dosage group of capsule and GUSHUKANG group can improve maximum oar degree, energy absorption; Significant difference is all arranged.And the heavy dose of group of casting capsule significantly is being better than other treatment group aspect raising maximum load, maximum oar degree, the energy absorption.
Show that the casting capsule can obviously improve the mechanical property of osteoporosis rat osseous tissue, reduces fracture risk.This and the casting capsule bone density that can raise increase bone mineral content content, and it is relevant to increase the bone amount.
8, osseous tissue morphometry
Table 8 medicine is to the influence of rat bone tissue norphometry mathematic(al) parameter
Bone resorption surface, the agent bone trabecula surface average bone wall mineralising deposition group N in bone formation surface
Amount percentage ratio (%) percentage ratio (%) percentage ratio (%) thickness (μ m) (μ m/d)
55.7 ± 2.7
* *4.55 ± 0.67
* *0.44 ± 0.03
* *Sham operated rats-6 10.7 ± 1.1
* *4.03 ± 0.58
* *
△△
△△
△△△
35.3 ± 4.5 11.81 ± 1.71 21.1 ± 2.9 2.82 ± 0.34 0.24 ± 0.02 model group-6
△ △ △ △ △ △ △ △ △ △ △ △ △ △ △GUSHUKANG KELI 42.8 ± 5.7
*8.62 ± 0.91
* *16.3 ± 1.9
* *3.35 ± 0.36
*0.31 ± 0.03
* *
2.7 6 groups
△ △ △ △ △ △ △ △Casting capsule group 0.4 6 48.8 ± 4.5
* *6.64 ± 0.84
* *12.2 ± 1.6
* *3.85 ± 0.28
* *0.37 ± 0.03
* *
41.0 ± 3.5
*8.91 ± 1.27
* *16.8 ± 2.2
*3.27 ± 0.40 0.28 ± 0.03
*Casting capsule group 0.2 6
△△
△△
△△△
△
△△△
37.4 ± 3.9 10.59 ± 1.19 20.2 ± 2.3 3.10 ± 0.30 0.25 ± 0.03 casting capsule groups 0.1 6
△△△
△△△
△△△
△△
△△△
Annotate: 1. compare with model group,
* *P＜0.001;
*P＜0.01;
*P＜0.05.
2. compare with the heavy dose group,
△ △ △P＜0.001;
△ △P＜0.01;
△P＜0.05.
Experimental result shows that after the rat castration, bone trabecula and form thereof take place obviously to change, bone trabecula surface percentage, average bone wall thickness and mineralising deposition all significantly are lower than the blank group, bone formation surface percentage, bone resorption surface percentage obviously increase, and the bone trabecula decreased number attenuates, sparse, the bone trabecula gap increases, show the rat castration after, bone formation and bone resorption are all hyperfunction, but bone resorption is relatively greater than bone formation, causes bone loss and causes osteoporosis.
Casting big or middle dosage group of capsule and GUSHUKANG group can make bone trabecula surface percentage, mineralising deposition improve than model group, significantly reduce bone formation surface percentage, bone resorption surface percentage; Heavy dose of group of casting capsule and GUSHUKANG group can make average bone wall thickness increase; Significant difference is all arranged.The heavy dose of group of casting capsule all is being better than other dosage group aspect above-mentioned five indexs.
Show that the casting capsule can obviously promote the formation and the mineralising of osteoporosis rat osteoid, and suppress bone resorption, osteoplastic supernormal growth, can improve the microstructure of bone to a certain extent, thereby both increase the bone amount, improved bone structure again, therefore the mechanical property of bone has been improved greatly.
Experimental example 2:The casting capsule is to the influence of castrated rats bone formation morphosis
After the decalcification bone slice HE dyeing, as seen microscopically is observed, and casting capsule treatment group increases slightly than model group bone trabecula, and breakaway poing reduces, and bone trabecula bar number increases, and arranges more neat; Arrangement of collagen fibers is neat, and seriality is better.
Experimental example 3:The casting capsule is to the influence of retinoic acid osteoporosis rat experimental model
Grouping and administration: get 60 of rats.Be divided into 6 groups at random by body weight.Blank group (n=10), osteoporosis model group (n=10), positive control drug GUSHUKANG KELI group (n=10), the casting heavy dose of group of capsule (n=10), dosage group (n=10) in the casting capsule, casting capsule small dose group (n=10).It is 1.33% beta-schardinger dextrin-aqueous solution that blank group, model group rat give concentration every day, positive drug group rat gives GUSHUKANG KELI 2.7g/kg (for 8 times of the clinical consumption per day of adult) every day, and the large, medium and small dosage group of casting capsule rat is cast capsule dosage every day respectively and is respectively 400mg/kg, 200mg/kg, 100mg/kg (be respectively the clinical consumption per day of adult 8,4,2 times).Continuous 4 weeks of gastric infusion (continuing to use for two weeks behind the retinoic acid of promptly stopping using).
Modeling method: with 1% sodium carboxymethyl cellulose is solvent, and retinoic acid is mixed with 0.7% suspension, and 4 ℃ keep in Dark Place, standby.Other 6 groups of rats except that blank group all give 70mg/kg filling stomach with 0.7% retinoic acid suspension every day, stop using after continuous 2 weeks.
Observation index:
(3) (Hydroxyproline, HOP), the mensuration of creatinine: assay method is with experiment one for urinary hydroxyproline.
(4) bone densitometry: assay method is with experiment one.
(5) biomechanics of bone strength detection: assay method is with experiment one.
(6) bone inorganic element content: assay method is with experiment one.
(7) the vertebral tissue morphometry is measured:
Prepare the horizontal undecalcified bone slice of 5 μ m by experiment one method, observe, measure.
(8) the bone formation morphosis is observed: method is with experiment one.
Result of the test:
All (X ± SD) expression relatively adopts SAS statistical software generalized linear model to carry out covariance analysis to result of the test between group, have the standard of significance as difference with p＜0.05 with mean+SD.
N is the rat number.In the experimentation, because the toxicity of retinoic acid causes indivedual rats deaths.It is different when so the number of rat is with the test grouping in the result of the test.
9, body weight change
Table 9 the weight of animals change list
Group after 4 weeks of administration before the dosage administration
(g/kg) the blank group-200.1 ± 12.2 (10) 281.8 ± 17.9 (10) of (g (only)) (g (only))
* * △ △ △Model group-198.6 ± 10.2 (10) 204.0 ± 33.1 (8)
000GUSHUKANG KELI group 2.7 201.2 ± 12.3 (10) 284.8 ± 13.2 (9)
* * △ △ △Casting capsule group 0.4 201.6 ± 13.2 (10) 290.1 ± 20.8 (8)
* * △ △ △Casting capsule 0.2 203.6 ± 9.6 (10) 290.1 ± 9.9 (9)
* * △ △ △Casting capsule group 0.1 200.8 ± 11.7 (10) 286.6 ± 17.1 (9)
* * △ △ △
Annotate: 1. compare p＞0.05 between each group before the administration.
2. compare after the administration with before the administration,
* *P＜0.001.
3. compare with model group after the administration,
△ △ △P＜0.001; Compare with the heavy dose group,
000P＜0.001.
Last table shows: each organizes the preceding body weight there was no significant difference of administration, and each group has comparability.The toxicity of retinoic acid can cause that the rat body weight growth slows down even negative growth.And casting capsule and GUSHUKANG all can suppress the toxicity of retinoic acid, thereby make it to improve.
10, serum calcium, phosphorus, alkali phosphatase and urine proline/creatinine table 10 medicine are to the influence of rat model serum calcium, phosphorus, alkali phosphatase and urine proline/creatinine
Dosage N Ca P ALP group HOP/Cr
(g/kg) (only) (mmol/L) (mmol/L) (U/L) blank group-10 12.8 ± 1.6 7.41 ± 0.78 140.8 ± 17.3
* *0.86 ± 0.08
* * △ △Model group-8 11.8 ± 1.3 8.16 ± 1.14 107.3 ± 8.7
△ △ △2.40 ± 0.31
△ △ △GUSHUKANG KELI group 2.7 9 12.0 ± 1.6 8.14 ± 0.98 131.1 ± 15.0
* *1.31 ± 0.18
* * △Casting capsule group 0.4 8 11.8 ± 1.7 8.17 ± 1.18 138.6 ± 15.8
* *1.11 ± 0.10
* *Casting capsule group 0.2 9 11.9 ± 1.0 7.48 ± 1.00 121.3 ± 10.9
* △1.35 ± 0.15
* * △ △Casting capsule group 0.1 9 11.8 ± 0.9 7.22 ± 0.93 108.4 ± 8.9
△ △ △1.70 ± 0.17
* * △ △ △
Annotate: 1. compare with model group,
* *P＜0.001;
*P＜0.05.
2. compare with the heavy dose group,
△ △ △P＜0.001;
△ △P＜0.01;
△P＜0.05.
After test shows the rat oral gavage retinoic acid, compare with the blank group, can cause serum alkaline phosphatase to reduce, the ratio of urinary hydroxyproline and creatinine raises, and then causes bone formation to reduce, and bone resorption is hyperfunction.
Casting big or middle dosage group of capsule and GUSHUKANG can make alkaline phosphatase activities raise than model group; Casting large, medium and small dosage group of capsule and GUSHUKANG all can make urinary hydroxyproline/creatinine reduce than model group; Significant difference is all arranged.The effect of the heavy dose of group of casting capsule is significantly better than the other treatment group.
Show that the casting capsule can improve the serum alkaline phosphatase that retinoic acid causes and the ANOMALOUS VARIATIONS of urinary hydroxyproline/creatinine.
11, femoral bmd
Table 11 medicine influences group dosage (g/kg) N (only) BMD (g/cm to the rat model femoral bmd
2) blank group-10 0.1611 ± 0.0074
* * △Model group-8 0.1442 ± 0.0060
△ △GUSHUKANG KELI group 2.7 9 0.1521 ± 0.0050
*Casting capsule group 0.4 8 0.1545 ± 0.0090
*Casting capsule group 0.2 9 0.1496 ± 0.0058 casting capsule group 0.1 9 0.1456 ± 0.0063
△ △
Annotate: 1. compare with model group,
* *P＜0.001;
*P＜0.01;
*P＜0.05.
2. compare with the heavy dose group,
△ △ △P＜0.001;
△ △P＜0.01;
△P＜0.05.
Retinoic acid osteoporosis rat model group is compared bone density with normal group and is significantly reduced.Show that irritating the stomach retinoic acid can cause bone destruction.
The heavy dose of group of casting capsule, GUSHUKANG group can make bone density than the remarkable rising of model group, and significant difference is arranged.
Show that the casting capsule can resist the rat femur bone density reduction that filling stomach retinoic acid causes.
12, femur inorganic elements
Table 12 medicine is to the influence of rat femur inorganic element content
Dosage N Ca P Zn Mg group
(g/kg) (only) (mg/g) (mg/g) blank group-10 198.4 ± 5.4 of (mg/g) (μ g/g)
* *115.6 ± 7.1 237.9 ± 15.1
* *3.99 ± 0.16
* *Model group-8 176.2 ± 4.3
△ △ △115.2 ± 3.8 216.1 ± 3.1
△ △ △3.72 ± 0.12
△ △ △GUSHUKANG KELI group 2.7 9 188.6 ± 5.9
* * △115.5 ± 4.2 245.7 ± 11.8
* *3.85 ± 0.12
* △Casting capsule group 0.4 8 195.1 ± 6.0
* *116.7 ± 3.7 239.4 ± 9.8
* *3.97 ± 0.12
* *Casting capsule group 0.2 9 188.4 ± 3.3
* * △ △115.4 ± 2.8 235.1 ± 13.8
*3.82 ± 0.08
△Casting capsule group 0.1 9 182.2 ± 5.2
* △ △ △114.7 ± 3.2 234.3 ± 10.5
*3.77 ± 0.10
△ △ △
Annotate: 1. compare with model group,
* *P＜0.001;
*P＜0.01;
*P＜0.05.
2. compare with the heavy dose group,
△ △ △P＜0.001;
△ △P＜0.01;
△P＜0.05.
Osteoporosis rat due to the retinoic acid, the content of part inorganic elements also can significantly reduce in the osseous tissue.
Casting large, medium and small dosage group of capsule and GUSHUKANG group can obviously improve the content of bone calcium, zinc; Heavy dose of group of casting capsule and GUSHUKANG group can obviously improve the content of bone magnesium; Significant difference is all arranged.The effect that the heavy dose of group of casting capsule improves bone calcium, bone magnesium obviously be better than GUSHUKANG and wherein, low dose.
Show that casting capsule loses the rat bone tissue inorganic elements that filling stomach retinoic acid causes, and improves significantly.
13, femur biomechanics
Table 13 medicine is to the influence of rat femur biomechanical strength
The maximum oar degree of dosage N maximum load bending rigidity energy absorption group
(g/kg) (only) (N) (mm) (N/mm) (Nmm) blank group-10 95.4 ± 11.0
* *1.01 ± 0.11
* *176.8 ± 16.0
* *25.2 ± 2.2
* *Model group-8 74.1 ± 8.3
△ △ △0.74 ± 0.10
△ △ △150.7 ± 13.5
△ △13.9 ± 1.5
△ △ △GUSHUKANG KELI group 2.7 9 84.6 ± 9.5
* △0.87 ± 0.10
* △166.6 ± 15.3
*21.7 ± 2.6
* * △Casting capsule group 0.4 8 93.7 ± 7.9
* *0.98 ± 0.09
* *175.1 ± 14.6
*24.1 ± 1.9
* *Casting capsule group 0.2 9 83.4 ± 3.8
* △0.79 ± 0.08
△ △ △162.4 ± 15.7 20.1 ± 2.1
* * △ △ △Casting capsule group 0.1 9 80.9 ± 10.0
△ △0.76 ± 0.08
△ △ △153.8 ± 13.8
△ △16.9 ± 2.2
* △ △ △
Annotate: 1. compare with model group,
* *P＜0.001;
*P＜0.01;
*P＜0.05.
2. compare with the heavy dose group,
△ △ △P＜0.001;
△ △P＜0.01;
△P＜0.05.
The result shows that retinoic acid can cause rat femur biomechanics index significantly to reduce, and increases the danger of fracture.
Casting big or middle dosage group of capsule and GUSHUKANG can improve maximum load; Heavy dose of group of casting capsule and GUSHUKANG group can improve maximum oar degree, bending rigidity, and casting large, medium and small dosage group of capsule and GUSHUKANG can improve energy absorption; Significant difference is all arranged.And the heavy dose of group of casting capsule significantly is being better than other treatment group aspect raising maximum load, maximum oar degree, the energy absorption.
Show that the casting capsule can obviously improve the mechanical property of retinoic acid osteoporosis rat osseous tissue, reduces the danger of fracture.
14, bone histomorphometry
Table 14 medicine is to the influence of rat bone tissue norphometry mathematic(al) parameter
The average bone wall of agent bone trabecula area bone resorption area bone formation area mineralization rate group N
Amount percentage ratio (%) percentage ratio (%) percentage ratio (%) thickness (μ m) (μ m/d)
42.5 ± 4.2
* *19.48 ± 1.37
* *0.37 ± 0.05
* *Blank group-10 4.79 ± 0.55
* *4.69 ± 0.65
* *
△
△△△
△△
29.2 ± 3.3 18.54 ± 2.50 5.05 ± 0.70 2.96 ± 0.39 0.16 ± 0.02 model group-8
△ △ △ △ △ △ △ △ △ △ △ △ △ △ △GUSHUKANG 33.2 ± 2.3
*8.54 ± 0.85
* *13.48 ± 1.42
* *3.96 ± 0.21
* *0.26 ± 0.03
* *
2.7 9 groups
△ △ △ △ △ △ △ △The casting capsule
0.4 8 38.2 ± 3.8
* *5.69 ± 0.70
* *16.04 ± 2.02
* *4.55 ± 0.52
* *0.30 ± 0.04
* *Group casting capsule 32.5 ± 2.7 10.30 ± 1.23
* *11.44 ± 1.54
* *3.60 ± 0.48
*0.25 ± 0.03
* *
0.2 9 groups
△ △ △ △ △ △ △ △ △ △ △ △Casting capsule 30.3 ± 1.9 16.28 ± 2.16
*9.46 ± 1.33
* *3.07 ± 0.35 0.18 ± 0.02
0.1 9 groups
△ △ △ △ △ △ △ △ △ △ △ △ △ △ △
Annotate: 1. compare with model group,
* *P＜0.001;
*P＜0.01;
*P＜0.05.
2. compare with the heavy dose group,
△ △ △P＜0.001;
△ △P＜0.01;
△P＜0.05.
Behind the rat oral gavage retinoic acid, can cause bone formation to reduce, bone resorption is hyperfunction, destroys bone trabecula.
Compare with model group, heavy dose of group of casting capsule and GUSHUKANG group can increase the bone trabecula area percentage; Casting large, medium and small dosage group of capsule and GUSHUKANG group all can increase the bone formation area percentage, reduce the bone resorption area percentage.Casting big or middle dosage group of capsule and GUSHUKANG group can increase bone wall thickness, mineralising deposition.The every index of the heavy dose of group of casting capsule all is better than other treatment group.
Show that the casting capsule can resist the bone formation that retinoic acid causes and reduce, absorbs hyperfunction, the reparation bone trabecula.
15, osseous tissue morphosis:
Casting capsule treatment group increases slightly than model group bone trabecula, and breakaway poing reduces, and bone trabecula bar number increases, and arranges more neat; Arrangement of collagen fibers is neat, and seriality is better.(referring to appendix VII～VIII page or leaf coloured picture) experimental example 3: casting capsule experiment of analgesic research (writhing method)
Grouping and administration: animal is divided at random: the blank group; Positive drug group (the Rhizoma Corydalis sheet that relieves the pain, dosage 0.75g/kg are 10 times of the clinical consumption per day of being grown up); Cast capsule heavy dose (500mg/kg), middle dosage (250mg/kg), low dose of (125mg/kg) 3 dosage groups, be equivalent to 10,5,2.5 times of clinical people's consumption per day respectively; Every group 14, male and female half and half.
Mouse stomach medicine, matched group give equal capacity beta-schardinger dextrin-aqueous solution (concentration 0.83%), 1 time on the 1st, continuous 7 days.1 hour lumbar injection 0.6% acetic acid after the administration on the 7th, 0.2ml/ are only.Write down every mice and incubation period of writhing response occurs, and turn round the body number of times in 0～10 minute, 10～20 minutes, 20～30 minutes.
Result of the test: result of the test is all with mean+SD (X ± SD) expression.N is a number of animals.Relatively adopt SAS statistical software generalized linear model to carry out covariance analysis between group, have the standard of significance with p＜0.05 as difference.(the results are shown in Table 15)
Table 15 casting capsule Dichlorodiphenyl Acetate causes the influence of mouse writhing reaction
The dosage body weight is turned round body number of times (inferior) group N incubation period
0～10 minute 10～20 minutes 20～30 minutes matched group-14 22.7 ± 2.01 8.66 ± 5.90 5.78 ± 6.53 11.0 ± 8.32 10.57 ± 10.54 Rhizoma Corydalis of g/kg (g) (branch) sheet group 0.75 14 23.23 ± 2.19 19.19 ± 8.83 that relieve the pain
*0.36 ± 0.89
*2.86 ± 4.10
*3.86 ± 4.64
*Casting capsule group 0.5 14 22.44 ± 2.02 18.29 ± 10.18
*0.71 ± 1.53
*2.86 ± 4.79
*2.21 ± 2.54
*Casting capsule group 0.25 14 23.43 ± 1.91 18.18 ± 10.19
*1.86 ± 3.48 4.71 ± 9.25 6.43 ± 12.50 casting capsule groups 0.125 14 23.17 ± 1.57 12.17 ± 7.24 2.93 ± 5.09 9.07 ± 9.42 8.0 ± 7.14
Annotate: compare with model group:
*P＜0.01;
*P＜0.05.
Result of the test shows that the heavy dose of group of casting capsule, middle dosage group and positive drug group all can suppress the mouse writhing reaction that lumbar injection acetic acid causes to some extent.Show as the casting big or middle dosage group of capsule and positive drug group and can prolong writhing response incubation period, heavy dose of group of casting capsule and GUSHUKANG can reduce in the unit interval animal and turn round the body number of times; Significant difference is all arranged.Prompting casting capsule and the Rhizoma Corydalis sheet that relieves the pain all has obvious analgesic activity.
Experimental example 4:Casting capsule anti-inflammatory experimentation
Grouping and administration: 50 of rats are used in experiment, be divided into matched group (irritating the beta-schardinger dextrin-aqueous solution of stomach equal-volume 1.33%), dexamethasone treatment group (filling stomach dexamethasone 0.1mg/kg at random, be equivalent to 8 times of people's consumption per day), the large, medium and small dosage group of casting capsule (irritates stomach casting capsule 400mg/kg, 200mg/kg and 100mg/kg respectively, be equivalent to 8,4,2 times of clinical people's consumption per day respectively), 10 every group.Successive administration 3 days, last administration modeling after 30 minutes.
Modeling method: after the last administration 30 minutes, measure its left back sufficient sole of the foot volume and record earlier.Subsequently respectively only at the left back sufficient plantar subcutaneous injection 1% carrageenin suspension 0.1ml/ of each group rat.Surveyed once left back sufficient sole of the foot volume and record, continuous measurement 6 times subsequently every 1 hour.The record result, and be calculated as follows swelling rate and suppression ratio.
Result of the test shows
The big or middle dosage group of casting capsule can significantly suppress the pedal swelling due to the rat injection carrageenin, and heavy dose of group relatively has significant difference (p＜0.05, p＜0.01) at modeling 1 hour～4 hours, middle dosage in modeling 1 hour～3 hours and matched group.Casting capsule small dose group also has certain inhibition trend.
Experimental example 5:The casting capsule is to the experimentation of immune function of mice influence
1, the casting capsule is to the influence of mouse spleen lymphocyte propagation
60 Balb/C mices are divided into 5 groups at random, are respectively the normal control group; Polyactin group, dosage are 0.03mg/kg; Casting capsule group, dosage is respectively 500mg/kg (being equivalent to 10 times of people's consumption per days), 250mg/kg (being equivalent to 5 times of people's consumption per days), 125mg/kg (being equivalent to 2.5 times of people's consumption per days).Be administered once every day, and continuous 10 days, the normal control group gave the beta-schardinger dextrin-aqueous solution of equal volume 0.83%.
The result shows: the large, medium and small dosage of casting capsule all can significantly strengthen the propagation of normal mouse spleen T, bone-marrow-derived lymphocyte, compares (p＜0.05, p＜0.01) with matched group.Positive control drug polyactin group can significantly strengthen the propagation (p＜0.01) of normal mouse spleen T, bone-marrow-derived lymphocyte.
2, cast the influence of capsule to the mice serum agglutinin:
Grouping and administration: with 1.
The result shows: the big or middle dosage of casting capsule all can significantly improve the content of agglutinin in the normal mouse serum, compares (p＜0.05, p＜0.01) with matched group.Low dose also has certain reinforced effects.Three dosage groups of casting capsule have dose-effect relationship preferably.
3, the casting capsule is to the influence of mice delayed hypersensitivity
Grouping Yu Give medicine: 72 Balb/c mices are divided into normal control group (irritating the beta-schardinger dextrin-aqueous solution that stomach is given equal-volume 0.83%) at random; DTH model group (irritating the beta-schardinger dextrin-aqueous solution that stomach is given equal-volume 0.83%); Polyactin group (irritate stomach and give 0.03mg/kg); Casting capsule group, gastric infusion, dosage are respectively 125mg/kg, 250mg/kg, 500mg/kg (be equivalent to respectively clinical people's consumption per day 2.5,5,10 times), successive administration 8 days.
The result shows: the normal matched group of DTH control group mice auris dextra significantly thickens, and shows that the DTH model prepares successfully.The large, medium and small dosage group of casting capsule all can significantly strengthen dinitrofluorobenzene induced mice delayed hypersensitive reaction, compares p＜0.01 with the DTH matched group.And dose-effect relationship is preferably arranged.
The following example all can be realized the effect of above-mentioned experimental example.Embodiment 1:
Get Folium Epimedii 4000g and decoct with water three times, each 1 hour, add 20 times of water gagings for the first time, second and third time respectively adds 16 times of water gagings, and decocting liquid merges, and filters, filtrate is concentrated into the clear paste that relative density is 1.04 (60 ℃), adds ethanol and makes that to contain alcohol amount be 70%, leaves standstill 24 hours, get supernatant and reclaim ethanol to there not being the alcohol flavor, add water to medicinal liquid: medical material is 2: 1, adds hydrochloric acid to pH=3, cold preservation was placed 72 hours, filtered, precipitation is dry, and is standby; Filtrate is refining with the polyamide absorption method, polyamide column absorption method condition: blade diameter length ratio is 1: 3, and liquor strength is by medical material: medicinal liquid is 1: 2, pH=3, than applied sample amount by medical material: polyamide is 2.5: 1, wash 4 times of column volumes, 70% ethanol elution is to the eluent color burn, begin to collect eluent, collect 1 times of column volume, reclaim ethanol, concentrated, dry; Wash 7 times of column volumes, behind the eccysis ethanol, repeat sample; The polyamide renovation process: repeat behind the sample 3 times regeneration 1 time, adsorb impurity with 3 times of column volume eccysis of 0.5% sodium hydrate aqueous solution after, wash with the pH=3 aqueous hydrochloric acid solution, to effluent pH=3, repeat sample; Renewable 2 times of polyamide is gone up sample 9 times altogether; The per kilogram polyamide can be made with extra care 22.5 kilograms of Herba Epimedii; 70% ethanol elution, eluent reclaims ethanol, and being concentrated into relative density is the thick paste of 1.20～1.25 (60 ℃), drying.The dried cream of two parts is merged, pulverize, get Herba Epimedii extract, standby.Get Concha Ostreae 220g, 60 mesh sieves are crossed in pulverizing, add the acetic acid of 8 times of amounts 30%, and heating hydrolysis 4 hours filters, and filtrate decompression is concentrated into semisolid, and drying is pulverized, and gets the Concha Ostreae hydrolysate, and is standby.Above-mentioned two parts are mixed, granulate, incapsulate, make 1000, promptly.
Embodiment 2:
To contain and surpass 50% Herba Epimedii total flavones or contain the Herba Epimedii effective part extract 167g that surpasses 15% icariin, contain the Concha Ostreae effective site hydrolysate 333g that surpasses 75% calcium acetate, mix, granulate, incapsulate, make 1000, promptly.
Claims (10)
1, a kind of pharmaceutical composition for the treatment of osteoporosis is characterized in that this pharmaceutical composition made by following crude drug:
Herba Epimedii: Concha Ostreae 15～25: 1～1.5.
2, the pharmaceutical composition of treatment osteoporosis as claimed in claim 1 is characterized in that this pharmaceutical composition made by following crude drug:
Herba Epimedii: Concha Ostreae is 20: 1.
3, a kind of pharmaceutical composition for the treatment of osteoporosis is characterized in that this pharmaceutical composition made by following crude drug:
The extract of Herba Epimedii effective site: the hydrolysate of Concha Ostreae effective site is 1～2: 1～3.
4, the pharmaceutical composition of treatment osteoporosis as claimed in claim 3 is characterized in that this pharmaceutical composition made by following crude drug:
Contain the extract that surpasses 50% Herba Epimedii total flavones or contain the Herba Epimedii effective site that surpasses 15% icariin: the hydrolysate that contains the Concha Ostreae effective site that surpasses 75% calcium acetate is 1: 2.
5,, it is characterized in that this pharmaceutical composition can directly or add excipient and make clinical acceptable forms as claim 1,2,3 or 4 described pharmaceutical compositions.
6, pharmaceutical composition as claimed in claim 5, the dosage form that it is characterized in that this pharmaceutical composition is tablet, capsule, oral liquid, granule, effervescent, buccal tablet.
7, the preparation of drug combination method of treatment osteoporosis as claimed in claim 1 or 2 is characterized in that this method is:
Get Folium Epimedii and decoct with water 2～4 times, each 1～2 hour, add 10～20 times of water gagings at every turn, decocting liquid merges, and filters, and filtrate is concentrated into the clear paste that relative density is 1.02～1.04 (60 ℃), add ethanol and make and contain alcohol amount and reach 60～80%, left standstill 12～48 hours, get supernatant and reclaim ethanol to there not being the alcohol flavor, add water to medicinal liquid: medical material is 2: 1, adds hydrochloric acid to pH=2～5, cold preservation, placed 24～72 hours, filter, precipitation is dry, and is standby; Filtrate is refining with the polyamide absorption method, polyamide column absorption method condition: blade diameter length ratio is 1: 2～5, and liquor strength is by medical material: medicinal liquid is 1: 1～3, pH=2～5, than applied sample amount is medical material: polyamide 2～5: 1, wash 3～6 times of column volumes, 60～80% ethanol elutions are to the eluent color burn, begin to collect eluent, collect 1～3 times of column volume, reclaim ethanol, concentrated, dry; Wash 5～10 times of column volumes, behind the eccysis ethanol, repeat sample; The polyamide renovation process: repeat behind the sample 2～5 times regeneration 1 time, adsorb impurity with 2～5 times of column volume eccysis of 0.5～1% sodium hydrate aqueous solution after, wash with pH=2～4 aqueous hydrochloric acid solutions, to effluent pH=2～4, repeat sample; Renewable 2 times of polyamide is gone up sample 9 times altogether; The per kilogram polyamide can be made with extra care 15～30 kilograms of Herba Epimedii; 60～80% ethanol elutions, eluent reclaims ethanol, and being concentrated into relative density is 60 ℃ of thick pastes of 1.20～1.25 down, drying; The dried cream of two parts is merged, pulverize, get Herba Epimedii extract, standby; Get Concha Ostreae, 60 mesh sieves are crossed in pulverizing, add the acetic acid of 8～12 times of amounts 20～30%, and heating hydrolysis 3～6 hours filters, and filtrate decompression is concentrated into semisolid, and drying is pulverized, and it is standby to get the Concha Ostreae hydrolysate; Above-mentioned two parts are mixed, pharmaceutical composition, can be directly or add excipient and make clinical acceptable forms with pharmaceutical composition.
8, the preparation of drug combination method of treatment osteoporosis as claimed in claim 7 is characterized in that this method is:
Get Folium Epimedii and decoct with water three times, each 1 hour, add 20 times of water gagings for the first time, second and third time respectively adds 16 times of water gagings, and decocting liquid merges, and filters, filtrate is concentrated into the clear paste that relative density is 1.04 (60 ℃), adds ethanol and makes and contain alcohol amount and reach 70%, leaves standstill 24 hours, get supernatant and reclaim ethanol to there not being the alcohol flavor, add water to medicinal liquid: medical material is 2: 1, adds hydrochloric acid to pH=3, cold preservation was placed 72 hours, filtered, precipitation is dry, and is standby; Filtrate is refining with the polyamide absorption method, polyamide column absorption method condition: blade diameter length ratio is 1: 3, and liquor strength is by medical material: medicinal liquid is 1: 2, pH=3, than applied sample amount by medical material: polyamide is 2.5: 1, wash 4 times of column volumes, 70% ethanol elution is to the eluent color burn, begin to collect eluent, collect 1 times of column volume, reclaim ethanol, concentrated, dry; Wash 7 times of column volumes, behind the eccysis ethanol, repeat sample; The polyamide renovation process: repeat behind the sample 3 times regeneration 1 time, adsorb impurity with 3 times of column volume eccysis of 0.5% sodium hydrate aqueous solution after, wash with the pH=3 aqueous hydrochloric acid solution, to effluent pH=3, repeat sample; Renewable 2 times of polyamide is gone up sample 9 times altogether; The per kilogram polyamide can be made with extra care 22.5 kilograms of Herba Epimedii; 70% ethanol elution, eluent reclaim ethanol, are concentrated into 60 ℃ of following relative densities and are 1.20～1.25 thick paste, drying; The dried cream of two parts is merged, pulverize, get Herba Epimedii extract, standby; Get Concha Ostreae, 60 mesh sieves are crossed in pulverizing, add the acetic acid of 8 times of amounts 30%, and heating hydrolysis 4 hours filters, and filtrate decompression is concentrated into semisolid, and drying is pulverized, and gets the Concha Ostreae hydrolysate, and is standby; Above-mentioned two parts are mixed, granulate, incapsulate.
9,, it is characterized in that this method is as the method for quality control of claim 1,2,3 or 4 described pharmaceutical compositions:
Differentiate: get aforementioned pharmaceutical compositions 0.2g, add each 1ml of sulphuric acid and ethanol, heat, the fragrance of ethyl acetate promptly takes place; Get aforementioned pharmaceutical compositions and pulverized 80 mesh sieves, take by weighing 0.05g, add ethanol 10ml, ultrasonic 4～8 minutes, filter, filtrate evaporate to dryness, residue add ethanol 1～2ml makes dissolving, as need testing solution; Other gets the icariin reference substance, adds methanol and makes the solution that every 1ml contains 0.1mg, in contrast product solution; According to thin layer chromatography, draw each 10 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, with 8～11: 0.8～1.2: 0.8～1.2: 0.8～1.2 ethyl acetate-butanone-formic acid-water is developing solvent, launch, take out, dry, spray is with 8～12% sulphuric acid ethanol liquid, in 100～105 ℃ of heating 2～4 minutes; In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical yellow spotting;
Assay: icariin is according to high effective liquid chromatography for measuring; Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler; 26～30: 70～74 acetonitrile-waters are mobile phase; The detection wavelength is 270nm; Number of theoretical plate calculates by the icariin peak should be not less than 1500; The reference substance solution preparation: it is an amount of that precision takes by weighing the icariin reference substance, adds the reference substance solution that methanol is made 5 μ g/ml, promptly; The need testing solution preparation: get aforementioned pharmaceutical compositions and pulverized 80 mesh sieves, precision takes by weighing 0.1g, puts in the 100ml tool plug conical flask, accurate 40～60% ethanol, the 45～55ml that adds, close plug claims to decide weight, ultrasonic to dissolving, claim again to decide weight, supply the weight that subtracts mistake with 40～60% ethanol, shake up, filter, get subsequent filtrate 0.5ml, put in the 10ml measuring bottle, add methanol to scale, shake up, as need testing solution; Algoscopy: accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing, inject chromatograph of liquid, measure, promptly; Pharmaceutical composition of the present invention contains Herba Epimedii in icariin, must not be less than 50mg/g;
Calcium acetate is got aforementioned pharmaceutical compositions and pulverized 80 mesh sieves, and precision takes by weighing 0.5g and puts in the crucible, and is slowly blazing to carbonization fully, puts and is chilled to room temperature; Make complete ashing at 500～700 ℃ of blazing 3h again, put coldly, residue adds dilute hydrochloric acid 4～8ml makes dissolving, put in the 10ml measuring bottle, thin up shakes up to scale, the accurate 1ml diluent of drawing adds water 100ml, 0.05% methyl red indicator 1ml, drip 10% potassium hydroxide to solution to light yellow, continue to add 10ml again, add calcein 0.1g, with the titration of disodiumedetate volumetric solution, to the yellow-green fluorescence disappearance of solution, and be orange, promptly; Every 1ml disodiumedetate volumetric solution is equivalent to the calcium acetate (Ca (Ac) of 15.8mg
2); Pharmaceutical composition of the present invention contains Concha Ostreae must not be less than 0.5g/g in calcium acetate.
10, as claim 1,2, the application of 3 or 4 described pharmaceutical compositions in the osteoporotic medicine of preparation treatment.
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| CNB021494711A CN1276756C (en) | 2002-11-21 | 2002-11-21 | A pharmaceutical composition for treating osteoporosis, and its preparation method |
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| CNB021494711A CN1276756C (en) | 2002-11-21 | 2002-11-21 | A pharmaceutical composition for treating osteoporosis, and its preparation method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101780132B (en) * | 2009-01-17 | 2012-03-07 | 毕力夫 | Mongolian medicine for treating osteoporosis and preparation method thereof |
| CN102526113A (en) * | 2012-03-15 | 2012-07-04 | 北京亚东生物制药有限公司 | Medicinal composition for treating osteoporosis and preparation method thereof |
| CN105768094A (en) * | 2016-04-19 | 2016-07-20 | 长春康彼达科技有限公司 | Health-care food for increasing bone density and making method thereof |
| CN112415157A (en) * | 2020-11-24 | 2021-02-26 | 内蒙古祈蒙药业股份有限公司 | Quality control method of Anxiao six-ingredient granules |
-
2002
- 2002-11-21 CN CNB021494711A patent/CN1276756C/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101780132B (en) * | 2009-01-17 | 2012-03-07 | 毕力夫 | Mongolian medicine for treating osteoporosis and preparation method thereof |
| CN102526113A (en) * | 2012-03-15 | 2012-07-04 | 北京亚东生物制药有限公司 | Medicinal composition for treating osteoporosis and preparation method thereof |
| CN105768094A (en) * | 2016-04-19 | 2016-07-20 | 长春康彼达科技有限公司 | Health-care food for increasing bone density and making method thereof |
| CN112415157A (en) * | 2020-11-24 | 2021-02-26 | 内蒙古祈蒙药业股份有限公司 | Quality control method of Anxiao six-ingredient granules |
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| Publication number | Publication date |
|---|---|
| CN1276756C (en) | 2006-09-27 |
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