CN1679765A - Chinese medicine extract for preventing female involutional syndrome or osteoporosis - Google Patents
Chinese medicine extract for preventing female involutional syndrome or osteoporosis Download PDFInfo
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Abstract
A Chinese medicine for treating the climacteric syndrome and osteoporosis of woman is prepared from 6 Chinese-medicinal materials including epimedium, anemarrhena rhizome, phellodendron bark, Chinese angelica root, etc through decocting.
Description
Technical field: the present invention relates to medical technical field, is a kind of Chinese medicine extract that can be used for preventing and treating menopausal syndrome or osteoporosis.
Background technology: the prescription that Herba Epimedii, Rhizoma Curculiginis, the Rhizoma Anemarrhenae, Cortex Phellodendri, Radix Angelicae Sinensis and Radix Morindae Officinalis Six-element Chinese medicine are formed, it is smart to have the kidney warming benefit, and the dual regulation function of nourishing YIN to lower pathogenic fire is generally used for treating menopausal syndrome, infertility.Determined curative effect is widely used.But normally will take as decoction behind this decocting for Chinese herbal medicine, not only decoct inconvenience, and have defectives such as dose is big, quality standard instability.So far do not see about it is prepared into Chinese medicine extract and is used to prevent and treat osteoporotic report.
Summary of the invention:
The present invention is prepared into Chinese medicine extract with Herba Epimedii, Rhizoma Curculiginis, the Rhizoma Anemarrhenae, Cortex Phellodendri, Radix Angelicae Sinensis and Radix Morindae Officinalis, except can be used for treating the menopausal syndrome, also can be used for osteoporosis.
The preparation method of Chinese medicine extract of the present invention is as follows:
One, prescription and proportioning (weight)
Herba Epimedii 8-15 part
Rhizoma Curculiginis 8-15 part
Rhizoma Anemarrhenae 8-15 part
Cortex Phellodendri 8-15 part
Radix Angelicae Sinensis 8-15 part
Radix Morindae Officinalis 8-15 part
Two, operating procedure
1. preparation extracting solution
According to routine above-mentioned medical material is pressed proportioning and decoct extraction, get extracting solution with gradation after extracting solvent soaking.
Extract solvent and be selected from the water or the arbitrary proportion mixture of ethanol and water, preferred 60-70% ethanol, its consumption can be 6-15 times (weight/volume) of medical material, and preferred 8-10 doubly.
Medical material is used earlier and was extracted solvent soaking 1-3 hour, perhaps soaked overnight.After the bubble profit routinely heating extraction be no less than 2 times, extraction time is 1-3 hour, preferred 2 hours.Merge extractive liquid.
2. concentrated and purified
With said extracted liquid concentrating under reduced pressure, the concentrated solution purification by macroporous resin is used earlier deionized water rinsing routinely, discard eluent, reuse 30-70% aquiferous ethanol eluting, preferred 50% ethanol, collect ethanol elution, decompression and solvent recovery gets extract of the present invention to doing.
Said macroporous resin is selected from models such as AB-8, D-101, ZTC-1 and SP 905, preferred ZTC-1 and SP-905.
Extract of the present invention is analyzed after testing, and general flavone content is no less than 35% in icariin, and wherein icariin content is not less than 4%; Total alkaloid content is no less than 10% in berberine, and wherein content of berberine is no less than 2%; Total saponin content is no less than 10% in Sarsasapogenin; Curculigoside content is no less than 2%, and extract of the present invention meets in the medicine registration management way requirement to 5 class Chinese medicine extract effective part groups.
Through experiment, extract of the present invention also has tangible function of resisting osteoporosis, thereby can mix with pharmaceutically suitable carrier, is used for preparation control menopausal syndrome or osteoporotic Chinese medicine preparation.
The specific embodiment:
Now in conjunction with the embodiments, the present invention is explained in detail
Embodiment 1. preparations extract of the present invention is got: Herba Epimedii, Rhizoma Curculiginis, the Rhizoma Anemarrhenae, Cortex Phellodendri, Radix Angelicae Sinensis and each 10kg of Radix Morindae Officinalis medical material, add 480kg 70% ethanol, decoct and extract three times, each 2 hours, merge extractive liquid,, filter, it is 1.05g/ml (25 ℃) that filtrate is evaporated to its relative density for 60 ℃.Macroporous resin column (the wet resin 1kg that per kilogram medical material usefulness has been handled well) after making liquid after concentrating by activation processing makes the active component in the liquid be adsorbed by resin column, and discards effluent.Water eluting macroporous resin column discards eluent; Continue with 50% ethanol elution macroporous resin column, collect eluent, 60 ℃ of decompression and solvent recoveries get extract 2.35kg to doing.General flavone content counts 35.5% with icariin, and wherein icariin content is 4.64%; Total saponin content counts 13.9% with Sarsasapogenin; Total alkaloid content counts 15% with berberine, and wherein content of berberine is 2.37%; Curculigoside content is 2.56%.
Embodiment 2. gets Herba Epimedii 12kg, Rhizoma Curculiginis 12kg, Rhizoma Anemarrhenae 10kg, Cortex Phellodendri 10kg, Radix Angelicae Sinensis and each 8kg of Radix Morindae Officinalis medical material, adds 600kg water, decocts to extract three times, each 2 hours, merge extractive liquid, filters, and it is 1.08g/ml that filtrate is evaporated to its relative density for 60 ℃.Make liquid after concentrating by the macroporous resin column after the activation processing, the active component in the liquid is adsorbed by resin column, and discard effluent.Water eluting macroporous resin column discards eluent; Continue with 60% ethanol elution macroporous resin column, collect eluent, 60 ℃ of decompression and solvent recoveries get extract 2.303kg to doing.General flavone content counts 33.0% with icariin, and wherein icariin content is 4.16%; Total saponin content counts 15.8% with Sarsasapogenin; Total alkaloid content counts 10.5% with berberine, and wherein content of berberine is 1.19%; Curculigoside content is 2.58%.
Embodiment 3. gets Herba Epimedii 15kg, Rhizoma Curculiginis 15kg, Rhizoma Anemarrhenae 8kg, Cortex Phellodendri 8kg, Radix Angelicae Sinensis and each 7kg of Radix Morindae Officinalis medical material, adds 600kg 50% ethanol, decocts to extract three times, each 2 hours, merge extractive liquid, filters, and it is 1.02g/ml that filtrate is evaporated to its relative density for 60 ℃.Macroporous resin column (the wet resin 1kg that per kilogram medical material usefulness has been handled well) after making liquid after concentrating by activation processing makes the active component in the liquid be adsorbed by resin column, and discards effluent.Water eluting macroporous resin column discards eluent; Continue with 40% ethanol elution macroporous resin column, collect eluent, 60 ℃ of decompression and solvent recoveries get extract 2.412kg to doing.General flavone content counts 38.4% with icariin, and wherein icariin content is 4.41%; Total saponin content counts 11.5% with Sarsasapogenin; Total alkaloid content counts 10.8% with berberine, and wherein content of berberine is 2.16%; Curculigoside content is 2.34%.
The quality standard analytical method of extract of the present invention is as follows:
1. differentiate
(1) gets extract 0.1g of the present invention, add ethanol 1ml dissolving, as need testing solution.Test according to thin layer chromatography, draw need testing solution and contain each 10 μ l of icariin 10 μ g/ml reference substance solution, put respectively in being on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, upper strata liquid with n-butyl alcohol-acetic acid-water (4: 1: 5) is that developing solvent launches, take out, dry, put under the uviol lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance icariin chromatograph on, show identical kermesinus speckle.Illustrate and contain the medical material Herba Epimedii extract in the extract of the present invention.
(2) according to the thin layer chromatography test, draw above-mentioned need testing solution and each 10 μ l of the reference substance solution that contains curculigoside 10 μ g/ml, put respectively in being on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, upper strata liquid with n-butyl alcohol-acetic acid-water (4: 1: 5) is that developing solvent launches, take out, dry, spray is with 2% potassium ferricyanide-2% liquor ferri trichloridi (1: 1).In the test sample chromatograph, with the corresponding position of reference substance curculigoside chromatograph on, show identical blue spot.Illustrate and contain the medical material Rhizoma Curculiginis extract in the extract of the present invention.
(3) according to the thin layer chromatography test, draw above-mentioned need testing solution and each 10 μ l of the reference substance solution that contains berberine 10 μ g/ml, put respectively in being on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, upper strata liquid with n-butyl alcohol-acetic acid-water (4: 1: 5) is that developing solvent launches, take out, dry, put under the uviol lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance berberine chromatograph on, show identical yellow spotting.Illustrate and contain the medical material Cortex Phellodendri extract in the extract of the present invention.
(4) get extract 0.5g of the present invention, add ethanol 5ml dissolving, add hydrochloric acid 1ml, reflux was concentrated into about 3ml after 1 hour, added water 5ml, extracted with benzene 10ml jolting, volatilized extracting solution, and residue adds benzene 1ml makes dissolving, as need testing solution.Other gets the Sarsasapogenin reference substance, adds benzene and makes the solution that every 1ml contains 5mg, in contrast product solution.Test according to thin layer chromatography, drawing each 10 μ l of above-mentioned two kinds of solution, put respectively on silica gel g thin-layer plate, is that developing solvent launches, takes out, dries with benzene-acetone, spray is with the mixed liquor of 8% vanillin ethanol solution and sulfuric acid solution, and it is clear to be heated to the speckle colour developing at 100 ℃.In the test sample chromatograph, with the corresponding position of reference substance Sarsasapogenin chromatograph on, show the speckle of same color.Illustrate and contain the medical material Rhizoma Anemarrhenae extract in the extract of the present invention.
(5) get extract 0.1g of the present invention, add ethanol 1ml dissolving, as need testing solution.Other gets Radix Angelicae Sinensis control medicinal material 1g, adds 70% ethanol extraction, is evaporated to the about 1.05g/ml of relative density, and by macroporous resin column, it is negative to be eluted to the molish reaction with distilled water, uses 50% ethanol elution, till the no ferulic acid of thin layer detection.Concentrating under reduced pressure evaporate to dryness ethanol liquid obtains Radix Angelicae Sinensis control medicinal material extract; Get Radix Angelicae Sinensis control medicinal material extract 0.1g, add ethanol 1ml dissolving, in contrast product solution.Test according to thin layer chromatography, draw above-mentioned two kinds of solution and contain each 10 μ l of ferulic acid 10 μ g/ml reference substance solution, putting respectively on silica gel g thin-layer plate, is that developing solvent launches, takes out, dries with normal hexane-ethyl acetate (8: 2), puts under the uviol lamp (365nm) and inspects.In the test sample chromatograph, with control medicinal material extract, the corresponding position of reference substance ferulic acid chromatograph on, show the fluorescence speckle of same color.Illustrate that extract of the present invention contains the extract of medical material Radix Angelicae Sinensis.
(6) sample thief 0.1g adds ethanol 1ml dissolving, as need testing solution.Other gets Radix Morindae Officinalis control medicinal material 1g, adds 70% ethanol extraction, is evaporated to the about 1.05g/ml of relative density, and by macroporous resin column, it is negative to be eluted to the molish reaction with distilled water, uses 50% ethanol elution, to colourless.Concentrating under reduced pressure evaporate to dryness ethanol liquid obtains Radix Morindae Officinalis control medicinal material extract, gets Radix Morindae Officinalis control medicinal material extract 0.1g, adds ethanol 1ml dissolving, in contrast product solution.According to the thin layer chromatography test, draw each 10 μ l of above-mentioned two kinds of solution, put in silica gel G F respectively
254On the lamellae, be developing solvent, launch, take out, dry, put under the uviol lamp (254nm) and inspect with benzene-ethyl acetate-formic acid (8: 2: 0.1).In the test sample chromatograph, with the corresponding position of control medicinal material extract chromatograph on, show the fluorescence speckle of same color.Illustrate that extract of the present invention contains the medical material Radix Morindae Officinalis extract.
2. check: check the organic solvent residual thing with gas chromatography
Chromatographic condition and system suitability test: with SIMPLICITY-5 capillary column (30m * 0.32mm * 0.25 μ m); Fid detector; 60 ℃ of capillary tube initial temperatures, 180 ℃ of injector temperatures, 220 ℃ of detector temperatures.With high purity nitrogen (99.99%) is carrier gas, pressure 7psi before the post, split ratio 30: 1.
Survey residual: ethanol, normal hexane, benzene, toluene, xylol, styrene, divinylbenzene
Interior mark: ethylo benzene
Solvent: N dinethylformamide
Measure: sample size 1 μ l.The sample introduction analysis begins 60 ℃ of constant temperature 4min of capillary tube, and 20 ℃ of temperature programmings then/min to 90 ℃, and keep 0.1min; 40 ℃ of temperature programmings again/min to 200 ℃, and keep 0.5min.
It is residual that extract of the present invention is not measured normal hexane, benzene, toluene, xylol, styrene, divinylbenzene.Meet the pertinent regulations of in the medicine registration management way organic solvent residual quality testing in the macroporous resin being surveyed.
3. assay:
(1) total flavones
The standard solution preparation: precision takes by weighing icariin standard substance 5.00mg, puts in the 25.0ml measuring bottle, adds dissolve with methanol and is settled to scale, promptly gets 0.2mg/ml icariin titer.
The need testing solution preparation: precision takes by weighing extract 5.00mg of the present invention, with being settled to the 25.0ml measuring bottle behind the dissolve with methanol, shakes up.
Standard curve is drawn: accurate 0.2mg/ml icariin titer 0.4,0.8,1.2,1.6, the 2.0ml of drawing, put in the 10.0ml measuring bottle, add 0.1mol/L aluminum trichloride solution 1.0ml, with methanol constant volume to 10.0ml, 10min is placed in 70 ℃ of waters bath with thermostatic control, take out the back room temperature and place 15min, the 411nm wavelength is measured trap.With trap A value to standard substance concentration return linear equation: A=13.513C+0.0009, r=0.9998; The range of linearity: 0.008～0.04mg/ml.
Sample determination: the accurate sample liquid 1.5ml that draws, put respectively in the 10.0ml measuring bottle, add 0.1mol/L aluminum trichloride solution 1.0ml, with methanol constant volume to 10.0ml, 10min is placed in 70 ℃ of waters bath with thermostatic control, and take out the back room temperature and place 15min, be blank with reagent, with spectrophotography, measure absorbance at the 411nm wavelength.
Extract of the present invention is pressed dry product and is calculated, and general flavone content must not be less than 35% in icariin.
(2) icariin
With high effective liquid chromatography for measuring
Chromatographic condition and system suitability test: SHIMADZU high performance liquid chromatograph, LC-10ATVP constant flow pump, SPD-10Auv-vis detector; Chromatographic column is: Hypersil-RDS C18 (4.6mm*200mm); Mobile phase: methanol: water: glacial acetic acid=80: 70: 2, the detection wavelength is 270nm.
The standard solution preparation: it is an amount of that precision takes by weighing the icariin standard substance, adds mobile phase and make the solution that every 1ml contains 1mg.
The need testing solution preparation: precision takes by weighing extract 10mg of the present invention, adds dissolve with methanol, is settled to 10ml.
Sample determination: accurate respectively standard solution and each 20ul of need testing solution of drawing, inject chromatograph of liquid, measure.
This product is pressed dry product and is calculated, and contains icariin and must not be less than 4%.
(3) total alkaloids
Standard solution preparation: precision takes by weighing berberine hydrochloride 2.00mg and puts in the 10ml volumetric flask, adds acetic acid-sodium-acetate buffer (pH 5.75) and dissolves and be settled to scale, promptly gets the berberine hydrochloride standard solution of 0.2mg/ml.
The need testing solution preparation: precision takes by weighing and is settled to the 25.0ml measuring bottle after extract 50.00mg of the present invention dissolves with buffer solution, shakes up.
Standard curve is drawn: the accurate berberine hydrochloride titer 0.5 of drawing 0.2mg/ml, 0.9,1.3,1.7,2.1ml place the erlenmeyer flask of tool plug respectively, all be diluted to 4.0ml with buffer, add 0.0125% bromothymol blue indicator 2ml and 10ml buffer solution, shake up, add chloroform 10ml, build bottle stopper, fully vibration 2min moves in the separatory funnel, leaves standstill 20min, layering, collect chloroform layer, get the 5ml chloroform with same method and extract again once, combined chloroform liquid, add the 0.5g anhydrous sodium sulfate dehydration, with the retinue solvent blank is contrast, measures 420nm wavelength absorption degree, with trap A value standard substance concentration is returned, get linear equation: A=0.0006112C+0.1121, γ=0.9968; The range of linearity: 0.004～0.02mg/ml.
Sample determination: the accurate sample solution 1ml that draws, to put respectively in the volumetric flask, method is measured under the sighting target directrix curve item.
Extract of the present invention is pressed dry product and is calculated, and total alkaloid content must not be less than 10% in berberine.
(4) berberine
With high effective liquid chromatography for measuring
Chromatographic condition and system suitability test: SHIMADZU high performance liquid chromatograph, LC-10ATVP constant flow pump, SPD-10Auv-vis detector; Chromatographic column is: Hypersil-RDS C18 (4.6mm*200mm); Mobile phase: methanol: 0.02ml/L NaH
2PO
4: 0.1ml/L triethylamine=40: 60: 2; The detection wavelength is 345nm.
The standard solution preparation: it is an amount of that precision takes by weighing the berberine standard substance, adds mobile phase and make the solution that every 1ml contains 1mg.
The need testing solution preparation: precision takes by weighing extract 10mg of the present invention, adds dissolve with methanol, is settled to 10ml.
Sample determination: accurate respectively standard solution and each 20ul of need testing solution of drawing, inject chromatograph of liquid, measure.
Extract of the present invention is pressed dry product and is calculated, and contains berberine and must not be less than 2%.
(5) total saponins
The standard solution preparation: precision takes by weighing Sarsasapogenin 5.00mg, adds the chloroform dissolving, is settled to 25.0ml, gets 0.200mg/ml Sarsasapogenin standard solution.
Need testing solution preparation: precision takes by weighing extract 0.5000g of the present invention, adds 2mol/L hydrochloric acid 20ml reflux, extract, 3hr, divides 3 extractions with the 60ml chloroform, and the collection extract is recycled to driedly, and the reuse chloroform is settled to 50ml.The accurate chloroform extraction liquid 1.0ml that draws is settled to 10ml with the chloroform dilution, shakes up, and is standby.
Standard curve is drawn: accurate 0.200mg/ml Sarsasapogenin standard solution 0.4,0.8,1.2,1.6, the 2.0ml of drawing, put in the 10ml scale test tube, and dry up.Add 0.2ml 5% vanillin-glacial acetic acid and 0.8ml perchloric acid, vortex, in 70 ℃ of water bath with thermostatic control heating 20min, take out, cool off with frozen water immediately, add glacial acetic acid 9.0ml, shake up, the 590nm wavelength is measured trap, with trap A value standard substance concentration is returned, get linear equation: A=18.615C+0.0281, r=0.9996; The range of linearity: 0.0816～0.408mg/ml.
Sample determination: the accurate sample solution 2.0ml that draws, place 10ml scale test tube, dry up.Add 0.2ml 5% vanillin-glacial acetic acid and 0.8ml perchloric acid, vortex in 70 ℃ of water bath with thermostatic control heating 20min, takes out, and with the frozen water cooling, adds glacial acetic acid 9.0ml immediately, shake up, and be blank with reagent, according to spectrophotography, under the 590nm wavelength, measure trap.
Extract of the present invention is pressed dry product and is calculated, and total saponin content must not be less than 10% in Sarsasapogenin.
(6) curculigoside
With high effective liquid chromatography for measuring
Chromatographic condition and system suitability test: SHIMADZU high performance liquid chromatograph, LC-10ATVP constant flow pump, SPD-10Auv-vis detector; Chromatographic column is: Hypersil-RDS C18 (4.6mm*200mm); Mobile phase: methanol: water: glacial acetic acid=50: 80: 2; The detection wavelength is 283nm
The standard solution preparation: it is an amount of that precision takes by weighing the curculigoside standard substance, adds mobile phase and make the solution that every 1ml contains 1mg.
The need testing solution preparation: precision takes by weighing extract 10mg of the present invention, adds dissolve with methanol, is settled to 10ml.
Sample determination: accurate respectively standard solution and each 20ul of need testing solution of drawing, inject chromatograph of liquid, measure.
Extract of the present invention is pressed dry product and is calculated, and contains curculigoside and must not be less than 2%.
It is as follows that extract of the present invention is used for the pharmacodynamic experiment of osteoporosis:
One, extract of the present invention is to the influence of removal ovary osteoporosis rat model
1. experiment material and method
1.1 animal SD cleaning level rat is female, at 3 monthly ages, purchases in west, Shanghai pul than triumphant laboratory animal company limited.
1.2 medical material and reagent are by the Chinese medicine extract of embodiment 1 preparation, XIANLING GUBAO, Tongjitang Pharmacy Co., Ltd., Guizhou's (lot number: 03070407); Nilestriol, Hualian Pharmaceutical Co., Ltd., Shanghai's (lot number: 031008); Serum Ca, P, alkaline phosphatase assay test kit are purchased in Shanghai Foxing Changzheng medical science Co., Ltd; Serum osteocalcin, calcitonin, estradiol are measured test kit and are purchased in Chinese Academy of Sciences Institute for Atomic Research.
1.3 instrument dual intensity X line borne densitometers, Tianjin, island universal testing machine, gamma-radiation calculating instrument.
1.4 experiment grouping and administration are divided into 7 groups at random with rat, 10 every group, comprise sham operated rats, model group, nilestriol group, XIANLING GUBAO group, extract low dose group of the present invention, middle dosage group, high dose group.Each organizes rat under 2% pentobarbital sodium (40mg/kg) anesthesia, and abdominal incision skin, muscle expose the abdominal cavity, find out bilateral ovaries, and sham operated rats keeps, all the other each group excisions.All animals are raised in 24～28 ℃ of draughty cleaning level Animal Houses, weigh once weekly.Sham operated rats, model group is irritated stomach and is given 0.5%CMC-Na, 5ml/kg/d, 6 times weekly, nilestriol group gastric infusion, 1mg/kg/ time, 1 time weekly; XIANLING GUBAO group gastric infusion 270mg/kg/d, 6 times weekly; The basic, normal, high dosage group of extract of the present invention gastric infusion is respectively 54mg/kg/d, 108mg/kg/d, 216mg/kg/d, 6 times weekly.Successive administration 3 months.
1.5 the serum biochemistry index determining is with rat anesthesia, eye socket is got blood, and separation of serum is measured serum calcium, content of inorganic phosphorus, alkaline phosphatase activities and estradiol, Bone Gla protein, calcitonin content.
1.6 next day after the administration of bone biomechanical index determining last, put to death rat, peel off its left side femur rapidly, behind length, minor axis width and the major axis width with vernier caliper measurement left side femur, carry out three point bending test with Tianjin, island universal testing machine, measure the biomechanical parameter of left side femur.
1.7 put to death rat next day after the administration of bone densitometry last, peels off its right side femur rapidly, measures the bone density of right side femur with dual intensity X line borne densitometers.
1.8 average relatively adopts variance test between the statistical procedures group.
2. experimental result
2.1 extract of the present invention is to the influence (the results are shown in Table 1) of removal ovary rat uterus weight and serum estradiol level
By table 1 as seen, after the ovariectomized rats, developing womb is suppressed, and alleviates 62% with sham operated rats comparison uterus weight, has significant difference.Nilestriol increases the uterus weight of removal ovary rat, relatively has significant difference with model group.XIANLING GUBAO and extract of the present invention all make the uterus weight of removal ovary rat increase.But compare there was no significant difference with model group.Model group rat blood serum estradiol content has descended 40% than sham operated rats, and each administration group all makes removal ovary rat blood serum estradiol content increase, and compares there was no significant difference with model group.Illustrate that extract of the present invention does not have remarkable influence to removal ovary rat uterus weight and serum estradiol level.
Table 1 extract of the present invention is to the influence of removal ovary rat uterus weight and serum estradiol level
(n＝10，Mean±SD)
| Uterus weight (g) | Estradiol (pg/ml) | |
| Dosage group high dose group in the sham operated rats model group nilestriol group XIANLING GUBAO group low dose group | ??0.736±0.229 ??0.280±0.114 ▲????0.713±0.327 △????0.342±0.056 ??0.292±0.034 ??0.310±0.155 ??0.397±0.227 | ?50.81±35.10 ?30.59±13.88 ?49.39±30.34 ?46.88±38.58 ?38.99±17.44 ?46.22±28.54 ?48.47±25.99 |
▲: compare P＜0.05 with sham operated rats; △: compare P＜0.05 with model group
2.2 extract of the present invention is to the influence (the results are shown in Table 2) of removal ovary rat blood serum biochemical indicator
By table 2 as seen, the content there was no significant difference of phosphorus in the serum between removal ovary and each the administration group rat.Model group and sham operated rats compare, and serum calcium levels does not have significance to change, but each medicine group all can significantly increase the level of removal ovary osteoporosis rat serum calcium.After the ovariectomized rats, alkali phosphatase significantly raises, and nilestriol and XIANLING GUBAO can reduce the serum alkaline phosphatase rising that removal ovary causes, but compare there was no significant difference with model group.The extract of the present invention of various dose all can reduce the rising of the serum alkaline phosphatase that removal ovary causes, high dose group and model group relatively have significant difference.Behind the removal ovary, serum osteocalcin content raises, and relatively has significant difference with sham operated rats.Nilestriol can reduce the rising of the serum osteocalcin that removal ovary causes to a certain extent, XIANLING GUBAO can significantly increase the level of removal ovary rat blood serum Bone Gla protein, and the extract of various dose does not have the influence of significance to the level of removal ovary rat blood serum Bone Gla protein.After the ovariectomized rats, the calcitonin level is kept normally in the blood, and each medicine group does not make significant difference to the level of removal ovary rat blood serum calcitonin.Illustrate that extract of the present invention can suppress the high conversion of bone that removal ovary causes.
Table 2 extract of the present invention is to the influence of removal ovary rat blood serum biochemical indicator
(n＝10，Mean±SD)
| Phosphorus (mmol/L) | Calcium (mmol/L) | Alkali phosphatase (U) | Bone Gla protein (ng/ml) | Calcitonin (pg/ml) | |
| Dosage group high dose group in the sham operated rats model group nilestriol group XIANLING GUBAO group low dose group | 1.74±0.33 1.56±0.11 2.23±0.30 △1.85±0.36 1.75±0.46 1.48±0.15 1.51±0.18 | 2.83±0.38 2.57±0.30 4.20±0.68 △4.64±1.83 △3.56±1.08 △5.54±1.59 △5.74±1.87 △ | 81.16±20.15 110.03±23.91 ▲100.17±37.95 98.76±24.69 111.00±24.57 96.11±17.45 85.48±24.97 △ | 0.87±0.33 1.41±0.45 ▲1.25±0.41 2.46±0.40 △1.60±0.34 1.32±0.23 1.48±0.51 | 50.90±7.67 47.86±12.06 72.06±18.08 43.97±5.31 46.76±7.96 46.10±4.69 44.87±6.17 |
▲: compare P＜0.05 with sham operated rats; △: compare P＜0.05 with model group
2.3 extract of the present invention is to the influence (the results are shown in Table 3) of removal ovary rat bone biomechanics index
By table 3 as seen, after the ovariectomized rats, the maximum load of femur does not have significance to change, and nilestriol, XIANLING GUBAO and extract of the present invention do not have the influence of significance yet to the maximum load of femur.The extract of the present invention of the female alcohol of denier, XIANLING GUBAO and high dose all can significantly improve maximum defluxion, maximum strain and the energy absorption of removal ovary rat femur three point bending test.Illustrate that extract of the present invention can increase the collagen content of bone matrix, strengthen the ability of its anti-fracture.
Table 3 extract of the present invention is to the influence of removal ovary rat bone biomechanics index
(n＝10，Mean±SD)
| Maximum load (N) | Maximum defluxion (mm) | Maximum strain (%) | Energy (J) | |
| Dosage group high dose group in the sham operated rats model group nilestriol group XIANLING GUBAO group low dose group | 98.82±12.78 108.98±17.49 103.05±7.90 111.20±8.99 94.52±13.37 100.25±17.71 108.88±8.53 | 1.01±0.32 0.77±0.16 ▲1.08±0.27 △1.12±0.17 △0.80±0.24 0.97±0.34 1.09±0.18 △ | ??4.40±0.99 ??4.33±1.05 ??5.64±1.60 △????6.57±0.86 △????4.30±1.28 ??4.46±2.16 ??6.18±1.02 △ | ??0.0623±0.0116 ??0.0583±0.0154 ??0.0612±0.0220 ??0.0702±0.0131 △????0.0561±0.0194 ??0.0642±0.0159 ??0.0710±0.0204 △ |
▲: compare P＜0.05 with sham operated rats; △: compare P＜0.05 with model group
2.4 extract of the present invention is to the influence (the results are shown in Table 4) of removal ovary rat bone density
By table 4 as seen, after the ovariectomized rats, bone density significantly descends, and nilestriol and XIANLING GUBAO can significantly increase the bone density of removal ovary rat; The extract of the present invention of various dose all can be in various degree the bone density of raising removal ovary rat, bone density and the model group of high dose medicament group rat relatively have significant difference.Illustrate that extract of the present invention can improve the bone density of removal ovary rat, so have the osteoporotic effect of control.
Table 4 extract of the present invention is to the influence of removal ovary rat bone density
(n＝10，Mean±SD)
| Bone density (g/cm 2) | |
| Dosage group high dose group in the sham-operation group model group Nilestriol group Xianlinggubao group low dose group | ????0.2622±0.0097 ????0.2491±0.0078 ▲????????0.2630±0.0063 △????????0.2612±0.0032 △????????0.2497±0.0098 ????0.2509±0.0089 ????0.2602±0.0121 △ |
▲: compare P＜0.05 with sham operated rats; △: compare P＜0.05 with model group
Two, extract of the present invention is to osteoblast in neonatal calvaria cultures with by the effect of the inductive osteoclast of myelomonocyte.
1 experiment material and method
1.1 newborn Wistar rat is provided by the The 2nd Army Medical College Experimental Animal Center for animal and reagent; Trypsin and type i collagen enzyme are Worthington company product; Calf serum is a Hangzhou Ilex purpurea Hassk.[I.chinensis Sims biological engineering company limited product; MTT (tetramethyl azo tetrazolium bromide) is available from Shanghai biological engineering company limited; The RPMI-1640 culture medium is a Gibco company product, α-MEM culture medium, and newborn hyclone is Gibco company product; 1 α, 25-(OH)
2VitaminD
3, dexamethasone, naphthols AS-BI phosphate, six azo paramagenta are sigma company product; All available from Shanghai biochemical reagents company, glycol monoethyl ether, sodium potassium tartrate tetrahydrate etc. are homemade analytical reagent for paranitrophenol disodium hydrogen phosphate, paranitrophenol, diethanolamine.
1.2 cell culture is got newborn Wistar rat, under aseptic condition, separate cranium, reject periosteum and hetero-organization thereof,, osteocomma is cut into the fritter of 1mm * 1mm with eye scissors with D-Hank ' s liquid flushing 3 times, the trypsin of adding 0.25%, in 37 ℃ of digestion 30 minutes, abandon supernatant, D-Hank ' s flushing 3 times, the Digestive system that adds the type i collagen enzyme that contains 0.05% trypsin and 1mg/ml, 37 ℃ digested 1 hour, drew Digestive system, through 140 order nylon net filters, the bone relic of reuse RPMI-1640 culture fluid flushing digestion, collect flushing liquor, filter the back and mixes, centrifugal 10 minutes with 1000r/min with Digestive system, collecting cell, be fresh osteoblast, add the RPMI-1640 culture medium that contains 10% calf serum, adjusting density is 1 * 10
5Cell/ml is inoculated in the culture bottle of 100ml, puts 37 ℃, 5%CO
2Incubator in cultivate.Change liquid after 24 hours.The visual cell growing state changed liquid once in 2～3 days later on.By morphologic observation, HE dyeing and alkaline phosphatase staining are identified osteoblast.Medullary cell and osteoblast be suspended in jointly contain 10
-8M 1 α, 25 (OH)
2Vitamin D
3, 10
-7In the α of the dexamethasone of M and 10% hyclone-MEM culture medium, making osteoblastic concentration is 10
5Cell/ml, the concentration of myelomonocyte is 10
6Cell/ml is inoculated in the culture bottle, forms absorption lacuna with the tartaric-resistant of osteoclast with on cattle cortical bone osteocomma osteoclast is carried out form and Function Identification.
1.3 detecting, osteoblastic proliferation gets the osteoblast that the second filial generation is cultivated the end art, the trypsinization with 0.25%, and in containing the RPMI-1640 culture medium of 10% calf serum, adjusting cell concentration is 1 * 10 with cell suspension
5Cell/ml is inoculated in 96 well culture plates, every hole 100 μ l, and in 37 ℃, 5%CO
2Incubator in cultivate, after 24 hours, change the RPMI-1640 culture medium of 10% calf serum of the extract of the present invention that contains variable concentrations, and establish the negative control that does not contain medicine, continue to cultivate 48 hours, finish preceding 4 hours in cultivating, every hole adds 20 μ l MTT (5mg/ml), after cultivating end, abandon supernatant, every hole adds 150 μ l DMSO, places, formation De Jia Za granule is dissolved fully, survey absorption value in the 550nm place.
1.4 the osteoblast determination of alkaline phosphatase activity is got the second filial generation and is cultivated the osteoblast that finishes the end, the trypsinization with 0.25%, and in containing the RPMI-1640 culture medium of 10% calf serum, adjusting cell concentration is 1 * 10 with cell suspension
5Cell/ml is inoculated in 24 well culture plates, every hole 1ml, cultivated 24 hours, and behind the cell attachment, changed the RPMI-1640 culture medium of 10% calf serum of the extract of the present invention that contains variable concentrations, and establish the negative control that does not add medicine, and continue to cultivate 8 days, changed liquid 1 time in per 2 days.Behind the extract effect setting-up time of the present invention, with PBS washed cell 3 times, the diethanolamine 200 μ l that add 50mmol/L, 2.5mmol/L paranitrophenol disodium hydrogen phosphate 100 μ l, 37 ℃ were reacted 30 minutes, 0.3mol/L NaOH 100 μ l cessation reactions, get 150 μ l and add in 96 orifice plates, survey its absorbance in the 405nm place.Standard curve is the standard curve that the p-nitrophenyl phenol solution of variable concentrations is done in the absorption value and the concentration at 405nm place.
1.5 being suspended in osteoblast and myelomonocyte jointly, the detection of the anti-tartaic acid phosphatase of osteoclast positive cell contains 10
-81,25 (OH) of M
2Vitamin D
3With 10
-7In the α of the dexamethasone of M and 10% hyclone-MEM culture medium, making osteoblast concentration is 10
5Cell/ml, myelomonocyte concentration is 10
6Cell/ml is added to cell suspension in 96 well culture plates, and every hole 100 μ l are incubated at 37 ℃, 5%CO
2Incubator in, behind the cell culture 24h, change the α-MEM culture medium of the extract of the present invention that contains variable concentrations, do not add the negative control of medicine, contain 10 in the culture fluid
-81,25 (OH) of M
2Vitamin D
3With 10
-7The dexamethasone of M and 10% hyclone.After, changed liquid once in per 3 days.Cultivate after 10 days, carry out TRAP dyeing, every hole TRAP positive cell is counted.
1.6 the anti-tartaic acid phosphatase activity of osteoclast is measured and is cultivated osteoclast as previously mentioned, after 8 days, the osteoclast differentiation and maturation, change the α-MEM culture medium 100 μ l that contain variable concentrations extract of the present invention, and establish the negative control that does not contain medicine, continue to cultivate 48 hours, abandon supernatant, use the PBS washed twice, draw logical smudge cells with the district of 10 μ l 0.1%, add the reactant liquor (preparation of reactant liquor: take by weighing p-nitrophenyl disodium hydrogen phosphate 0.4g after 15 minutes, use deionized water dissolving, add Soluble tartar. 2.0g, be dissolved in water to 150ml, regulate PH to 3.5 with 1N HCl, add deionized water again to 200ml.) 100 μ l, in 37 ℃ of reactions 30 minutes, the sodium hydroxide solution cessation reaction that adds 100 μ l 1N rapidly, measure its absorption value in wavelength 410nm place, make standard curve with the p-nitrophenyl phenol solution, the nanomole numerical table of the paranitrophenol that generates with every hole shows the activity of anti-tartaic acid phosphatase.
2. result
2.1 influence (the results are shown in Table 5) to osteoblastic propagation and alkaline phosphatase activities
By table 5 as seen, extract of the present invention can concentration in the concentration range of 1-10 μ g/ml relies on ground increases osteoblastic propagation, but only under the mass action of 5 μ g/ml and 10 μ g/ml, osteoblastic propagation is compared with contrast has significant difference.Can concentration rely on ground increase osteoblast alkaline phosphatase activity in the concentration range of extract 2-10 μ g/ml of the present invention, and have significant difference compared with the control.Illustrate that extract of the present invention is to promote osteoplastic by increasing osteoblastic propagation and alkaline phosphatase activity.
Table 5. extract of the present invention is to the influence of osteoblast in neonatal calvaria cultures propagation and alkaline phosphatase activities
n＝6
| Extract concentrations | MTT measurement result (OD value) | Alkaline phosphatase activities (nmol/well) |
| 20 μ g/ml, 10 μ g/ml, 5 μ g/ml, 2 μ g/ml, 1 μ g/ml negative control | ????0.857±0.321 ????1.371±0.141 **????1.242±0.209 *????1.207±0.300 ????1.069±0.156 ????0.977±0.096 | ??142.62±17.9 ??178.68±5.92 **??174.31±5.09 **??165.41±5.48 *??151.27±17.21 ??152.78±4.64 |
Annotate: with the matched group ratio
*P＜0.05,
*P＜0.01
2.2 influence (the results are shown in Table 6) to formation, differentiation and the anti-tartaic acid phosphatase activity of osteoclast
By table 6 as seen, extract of the present invention dosage in the concentration range of 1-20 μ g/ml relies on the activity that ground suppresses formation, differentiation and the anti-tartaic acid phosphatase of osteoclast, but have only under the mass action of 5-20 μ g/ml, have significant difference compared with the control.The activity that formation, differentiation and the reduction anti-tartaic acid phosphatase of extract of the present invention by suppressing osteoclast be described reduces bone resorption.
Table 6. extract of the present invention is to the influence of formation, differentiation and the anti-tartaic acid phosphatase activity of osteoclast
n＝6
| Extract concentrations | TRAP positive cell number/hole | The activity of TRAP (nmol/well) |
| 20 μ g/ml, 10 μ g/ml, 5 μ g/ml, 2 μ g/ml, 1 μ g/ml negative control | ????200±10 **????248±19 **????276±20 **????354±20 ????351±11 ????364±14 | ????14.56±0.87 **????16.78±1.05 **????16.96±0.96 *????18.98±2.01 ????20.38±1.94 ????25.08±0.92 |
Annotate: with the matched group ratio
*P＜0.05,
*P＜0.01
Claims (3)
1. Chinese medicine extract, preparation method is as follows:
(1) prescription and proportioning (weight)
Herba Epimedii 8-15 part
Rhizoma Curculiginis 8-15 part
Rhizoma Anemarrhenae 8-15 part
Cortex Phellodendri 8-15 part
Radix Angelicae Sinensis 8-15 part
Radix Morindae Officinalis 8-15 part
(2) operating procedure
(1) preparation extracting solution
According to routine, above-mentioned medical material is pressed proportioning decoct extraction with gradation after extracting solvent soaking, get extracting solution, extract the arbitrary proportion mixture that solvent is selected from water or ethanol and water;
(2) concentrated and purified
Routinely with said extracted liquid concentrating under reduced pressure, the concentrated solution purification by macroporous resin, macroporous resin deionized water eluting, discard eluent, reuse 30-70% ethanol elution is collected ethanol elution, decompression and solvent recovery is to doing, get extract of the present invention, its general flavone content is not less than 35% in icariin, and wherein icariin content is not less than 4%; Total alkaloid content is not less than 10% in berberine, and wherein content of berberine is not less than 2%; Total saponin content is not less than 10% in Sarsasapogenin; Curculigoside content is not less than 2%.
2. by the described Chinese medicine extract of claim 1, it is characterized in that proportioning is each 10kg of Herba Epimedii, Rhizoma Curculiginis, the Rhizoma Anemarrhenae, Cortex Phellodendri, Radix Morindae Officinalis and Radix Angelicae Sinensis, with 70% ethanol serves as to extract solvent, decoct and extract 3 times, merge extractive liquid,, filter, being evaporated to the filtrate relative density under 60 ℃ is 1.05g/ml, with the macroporous resin ZTC-1 purification after the activation processing, behind the macroporous resin column water eluting, reuse 50% ethanol elution is collected ethanol elution, 60 ℃ of decompression and solvent recoveries get extract of the present invention to doing.
3. the application in preparation control menopausal syndrome or medicine for treating osteoporosis by claim 1 or 2 described Chinese medicine extract.
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