CN101474357B - Pharmaceutical composition for treating female barrenness - Google Patents
Pharmaceutical composition for treating female barrenness Download PDFInfo
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- CN101474357B CN101474357B CN 200910045722 CN200910045722A CN101474357B CN 101474357 B CN101474357 B CN 101474357B CN 200910045722 CN200910045722 CN 200910045722 CN 200910045722 A CN200910045722 A CN 200910045722A CN 101474357 B CN101474357 B CN 101474357B
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Abstract
The invention discloses a pharmaceutical composition for the treatment of female infertility as well as a preparation method and a quality control method thereof, the composition is any one of pharmaceutically acceptable preparations such as pellet, granule, tablet, syrup, mixture, oral liquid, dripping pill, capsule and injection prepared by epimedium brevicornum, sealwort, teasel root, astragalus root, rhizome cyperi and licorice. The quality control method thereof comprises the steps of: thin-layer identification of the epimedium brevicornum, sealwort, radix dipsaci, licorice, licorice and astragalus root; HPLC content measurement of the epimedium brevicornum, radix dipsaci and licorice; measurement of total contents of flavone and saponin. The pharmacodynamical experiments include: in vivo experiment, in vitro cell experiment and clinical experiment. The experiments demonstrate that: the inventive pharmaceutical composition has the effects of warming kidney, replenishing sperm and conditioning emmenia, and the pharmaceutical composition can be used for the treatment of the female infertility.
Description
Technical field
The present invention relates to a kind of pharmaceutical composition and preparation method thereof and method of quality control, particularly relate to a kind of pharmaceutical composition that is used for the treatment of female barrenness and preparation method thereof and method of quality control.
Background technology
It is generally acknowledged that the couple at child-bearing age lives together after marriage, not contraception, sexual life is normal, and the phenomenon that more than 2 years do not become pregnant in (U.S.'s department of obstetrics and gynecology teaching material and infertile association then are decided to be 1 year to the time) wife's side is referred to as " infertility ".The infertility or the infertility that are caused by bridegroom's or husband's side reason are called " male infertility " or " male sterility ", and common people generally are referred to as infertility with it.The female barrenness paathogenic factor is a lot, and pathogeny is rather complicated, and principal element is a maturation of ovum obstacle etc.The male infertility principal element is spermioteleosis obstacle and sperm transportation obstacle etc.
Doctor trained in Western medicine is to use drug-induced ovulation, salpingemphraxis reopening operation, salpingectomy and laparoscopic technique removing focus to treat at the main treatment means of female barrenness at present; Treatment means at male infertility comprises medicine and operation, and medicine mainly comprises gonadotropin releasing hormone (GnRH), promoting sexual gland hormone (FSH, HMG), chorionic-gonadotropin hormone (HCG), clomiphene etc.; Operative treatment comprises epididymis decline fixation, spermatic vein ligation, vasovasostomy and deferent duct-epididymis-testis anastomosis.Assisted Reproductive Technology ART comprises that injection (ICSI) also is the conventional means of treatment sterility and infertility in artificial insemination (IUI), in vitro fertilization-embryo transfer (IVF-ET), the monosperm endochylema in addition.Yet at present not only expense is expensive but also increase the weight of that patient suffering, cure rate are not high, recurrent is big for medicine and operative treatment, and side effect and sequela are big.In view of present Western medicine acts on single target spot or link mostly, cause unsatisfactory curative effect.And the modern Chinese medicine compound recipe is to treat this disease by many target spots, too many levels and multisystem, and to gynaecopathia, especially chronic and difficult miscellaneous disease has better curative effect, good help and excellent research object is provided for our research.
Summary of the invention
Technical problem to be solved by this invention provides a kind of evident in efficacy, the pharmaceutical composition of taking the treatment female barrenness that the back is difficult for recurrence and preparation method thereof and method of quality control.
In order to achieve the above object, the present invention realizes by the following technical solutions:
A kind of pharmaceutical composition that is used for the treatment of female barrenness, component is calculated by weight, and described pharmaceutical composition comprises following raw materials of effective components: Herba Epimedii 15-45 part, Rhizoma Polygonati 12-36 part, Radix Dipsaci 12-36 part, Rhizoma Cyperi 9-27 part, Rhizoma Acori Graminei 9-27 part, Radix Glycyrrhizae 6-18 part.
The pharmaceutical composition of described treatment female barrenness, component preferably includes following raw materials of effective components: 30 parts of Herba Epimedii, 24 parts of Rhizoma Polygonatis, 24 parts of Radix Dipsacis, 18 parts of Rhizoma Cyperis, 18 parts of Rhizoma Acori Graminei, 12 parts in Radix Glycyrrhizae by weight.
Described pharmaceutical composition is an acceptable forms on pill, granule, tablet, syrup, mixture, oral liquid, drop pill, capsule, any pharmaceutics of injection, and slow release or the application of controlled-release technology in above-mentioned dosage form.
Described pharmaceutical composition is preferably one or more in mixture, syrup, granule, capsule, the tablet.
Described pharmaceutical composition is preferably mixture.
The preparation method of the oral liquid of described pharmaceutical composition, Rhizoma Cyperi and Rhizoma Acori Graminei add 12 times of water gagings, distill and extract volatile oil in 6 hours, and the aqueous solution after distillation device is in addition collected; Medicinal residues add 12 times of water gagings again and decocted 1 hour, and medical filtration is standby; 4 remaining flavors decoct 3 times with 10 times of water gagings respectively, and each 2 hours, collecting decoction, filter, filtrate and above-mentioned medicinal liquid merge, and are evaporated to relative density 1.10-1.12/60 ℃, left standstill 24 hours, and got supernatant and reclaim ethanol and be concentrated into 1.04/20 ℃ of relative density, left standstill 48 hours, get supernatant and filter, filtrate adds aforementioned volatile oil, tween 80 and an amount of correctives, adjusts volume to 1000mL, filter, packing, sterilization promptly gets described oral liquid.
The preparation method of described pharmaceutical composition syrup, Rhizoma Cyperi and Rhizoma Acori Graminei add 12 times of water gagings, distill and extract volatile oil in 6 hours, and the aqueous solution after distillation device is in addition collected; Volatile oil becomes clathrate with the beta cyclodextrin enclose of 6 times of amounts; Medicinal residues add 12 times of water gagings again and decocted 1 hour, and medical filtration is standby; 4 remaining flavors decoct 3 times with 10 times of water gagings respectively, and each 2 hours, collecting decoction filtered, filtrate and above-mentioned medicinal liquid merge, and are evaporated to relative density 1.10-1.12/60 ℃, leave standstill 24 hours, get supernatant and reclaim ethanol and be concentrated into density 1.18, other gets sucrose and decocts with water for 65 parts, after the dissolving, filters, concentrate and make syrup,, boil with above-mentioned concentrated solution mixing, put coldly, add antiseptic, above-mentioned volatile oil, tween 80, adjust volume to 1000mL, packing, sterilization promptly gets described syrup.
The preparation method of described pharmaceutical composition tablet, Rhizoma Cyperi and Rhizoma Acori Graminei add 12 times of water gagings, distill and extract volatile oil in 6 hours, and the aqueous solution after distillation device is in addition collected; Volatile oil becomes clathrate with the beta cyclodextrin enclose of 6 times of amounts.Medicinal residues add 12 times of water gagings again and decocted 1 hour, and medical filtration is standby; 4 remaining flavors decoct 3 times with 10 times of water gagings respectively, each 2 hours, collecting decoction filters, and filtrate and above-mentioned medicinal liquid merge, be evaporated to the medicinal liquid of 1.10/60 ℃ of relative density, add ethanol and make and contain alcohol amount and reach 50%, stir, left standstill 24 hours, draw supernatant and reclaim ethanol, be condensed into relative density and be 1.33-1.35/60 ℃ extractum, get 1 part of extractum, add 2 times of amount sucrose, 1 times of amount dextrin and ethanol are an amount of, granulate, drying is with the beta cyclodextrin clathrate of volatile oil and the magnesium stearate mixing of 1wt%, tabletting promptly gets described tablet.
The preparation method of described pharmaceutical composition mixture with Herba Epimedii, Rhizoma Polygonati, Radix Dipsaci, Rhizoma Cyperi, Rhizoma Acori Graminei, Radix Glycyrrhizae Six-element, adds 8 times of water gagings and 4 times of water gagings decoction secondaries respectively, each back that decocts filters merging filtrate in 85 ℃ of insulations 20 minutes, being evaporated to relative density is not less than under 1.06/80 ℃, centrifugal, get supernatant, add 20 parts of sucrose, 0.3 part of sodium benzoate, 0.05 part of ethyl hydroxybenzoate, add water and make 1000mL, mixing, fill promptly gets described mixture.
The method of quality control of described pharmaceutical composition mixture comprises: the thin layer of Herba Epimedii, Rhizoma Polygonati, Radix Dipsaci, Rhizoma Cyperi, Rhizoma Acori Graminei, Radix Glycyrrhizae is differentiated; The HPLC assay of Herba Epimedii, Radix Glycyrrhizae, Radix Dipsaci; The assay of total flavones, total saponins.
The method of quality control of described pharmaceutical composition mixture, its pharmacodynamic study comprises:
(1) experiment in the body: the female rats endometriosis is duplicated in autotransplantation, detects blood plasma beta-endorphin β-EP, serum estradiol E
2, progesterone P level;
(2) cell in vitro experiment: cultivate gonad granulocyte, measure the pharmaceutical intervention growing state of cell afterwards.
The method of quality control of described mixture, its clinical experiment comprises: 60 routine patients are divided into treatment at random and organize 30 examples, matched group 30 examples, Drug therapy is after course of treatment, measure follicular diameter, measure anti-endometrial antibodies EMAb, measure TNF-a, interleukin-6 IL-6.
The method of quality control of pharmaceutical composition mixture of the present invention is specific as follows:
One, character
Brown to tan liquid, putting for a long time to have small amount of precipitate; Sweet, little hardship of distinguishing the flavor of.
Two, differentiate
(1) discriminating of Herba Epimedii
Get pharmaceutical composition mixture 10mL of the present invention, place polyamide column (polyamide 5g, internal diameter 0.9cm, column length 25cm) with the 100mL water elution, discard eluent,, collect eluent again with ethanol 100mL eluting, evaporate to dryness, residue add ethanol 2mL makes dissolving, as need testing solution; Other gets the Herba Epimedii reference substance, adds methanol and makes the solution that every 1mL contains 10ug, in contrast product solution; All components except that Herba Epimedii is made blank sample in the prescription ratio, with the method operation, as blank product solution.Draw each 4 μ L of above-mentioned 3 kinds of solution, put respectively on same silica gel g thin-layer plate, with ethyl acetate-butanone-formic acid-water (volume ratio 10: 1: 1: 1) be developing solvent, launch, take out, dry, spray, inspect putting under the ultra-violet lamp 365nm with the aluminum chloride test solution.Found that: in the test sample chromatograph, with the corresponding position of reference substance chromatograph on show the fluorescence speckle of same color, and negative controls does not have this speckle on the relevant position, i.e. explanation blank is noiseless.
(2) discriminating of Radix Glycyrrhizae
Get pharmaceutical composition mixture 10mL of the present invention, Dropwise 5 0wt% sulfuric acid solution 1mL, centrifugal, abandon supernatant, precipitation washes with water 2 times, and each 10mL gets precipitation, adds ethanol 10mL and makes dissolving, and is centrifugal, gets supernatant as need testing solution; Extracting liquorice acid ammonium reference substance adds ethanol and makes the solution that every 1mL contains 0.1mg, in contrast product solution; Other gets the negative control sample that lacks Radix Glycyrrhizae and makes negative control solution with the need testing solution preparation method.Draw each 10 μ L of above-mentioned three kinds of solution and put respectively on same silica GF254 lamellae, with ethyl acetate-formic acid-glacial acetic acid-water (volume ratio 15: 1: 1: 2) be developing solvent, launch, take out, dry.Put under the ultra-violet lamp 254nm and inspect.Found that: in the test sample chromatograph, with the corresponding position of reference substance chromatograph on show the speckle of same color, and negative controls does not have this speckle on the relevant position, i.e. explanation blank is noiseless.
(3) discriminating of Radix Dipsaci
Get pharmaceutical composition mixture 15mL of the present invention, with water saturated n-butanol extraction 3 times, each 15mL merges n-butyl alcohol liquid, and water bath method, residue add 3mL methanol makes dissolving, as need testing solution; Other gets the negative control sample that lacks Radix Dipsaci and makes negative control solution with method; Get Radix Dipsaci control medicinal material 1.2g, add water 30mL, decocting 15min filters, and gets filtrate, gets control medicinal material solution with legal system.Draw each 1 μ L of above-mentioned 3 kinds of solution, put respectively on same silica gel g thin-layer plate, (22: 40: 15: upper strata liquid 10) was as developing solvent with chloroform-ethyl acetate-methanol-water.Launch, take out, dry.Spray is with the ethanol solution of sulfuric acid of 10wt%, and 105 ℃ of heating to clear spot, are inspected under the daylight.Found that: in the test sample chromatograph, with the corresponding position of Radix Dipsaci control medicinal material chromatograph on show the speckle of same color, and negative controls does not have this speckle on the relevant position, i.e. explanation blank is noiseless.
(4) discriminating of Rhizoma Polygonati
Get pharmaceutical composition mixture 5mL of the present invention, put in the separatory funnel, with water saturation n-butanol extraction twice, each 10mL, merge extractive liquid, reclaims n-butyl alcohol to doing, and residue adds ethanol 2mL makes dissolving, as need testing solution; Other gets Rhizoma Polygonati medical material 4g, shines medical material solution in pairs with legal system.Drawing control medicinal material solution 1uL, need testing solution 4uL, put respectively on same silica gel g thin-layer plate, is developing solvent with benzene-ethyl acetate-water (volume ratio 5: 2: 0.1), launches, and takes out, and dries, and spray is with the 2,4 dinitrophenyl hydrazine alcoholic solution of 2wt%.Found that: in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color, and negative controls does not have this speckle on the relevant position, i.e. explanation blank is noiseless.
(5) discriminating of Rhizoma Cyperi
Get pharmaceutical composition mixture 10mL of the present invention, with water saturated n-butanol extraction 3 times, each 10mL merges n-butanol extracting liquid, reclaims n-butyl alcohol to doing, and residue adds ethanol 2mL makes dissolving, as need testing solution; Get scarce Rhizoma Cyperi self-control sample 10mL, make negative control solution with method; Other gets α-cyperone reference substance, adds ethyl acetate and makes the solution that every 1mL contains 1mg, in contrast product solution.Test according to thin layer chromatography, draw need testing solution, negative control solution each 5 μ L and reference substance solution 2 μ L, put respectively on same silica gel g thin-layer plate, with benzene-ethyl acetate-glacial acetic acid (volume ratio 60: 1: 1) is developing solvent, launch, take out, dry, put ultra-violet lamp and (inspect under the 254nm.Found that: in the need testing solution chromatograph, with the corresponding position of reference substance solution chromatograph on, show identical navy blue speckle, spray is placed a moment with the dinitrophenylhydrazine test solution, speckle fades to orange red, negative control is noiseless.
(6) discriminating of Rhizoma Acori Graminei
Get pharmaceutical composition mixture 10mL of the present invention, with water saturated n-butanol extraction 3 times, each 10mL, merge extractive liquid, reclaims n-butyl alcohol to doing, and residue adds ethanol 2mL makes dissolving, as need testing solution; Get scarce Rhizoma Acori Graminei self-control sample 10mL, make negative control solution with method; Other gets the Rhizoma Acori Graminei control medicinal material, adds petroleum ether and makes control medicinal material solution.According to the thin layer chromatography test, draw each 1 μ L of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with petroleum ether (60～90 ℃)-ethyl acetate (8: 2) is developing solvent, launches, and takes out, dry, placed about 1 hour, put under the ultra-violet lamp 365nm and inspect.Found that: in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color; Smoked clear with iodine vapor again to the speckle colour developing, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the speckle of same color.
Three, check
Meet " the every regulation under the Chinese pharmacopoeia mixture item.
Four, assay
1. the assay of glycyrrhizic acid
1.1 chromatographic condition
Chromatographic column: Zorbax Eclipse XDB-C18 post (5 μ m, 4.6mm * 150mm);
Mobile phase: methanol: 0.01mol/L hydrochloric acid solution (75: 25);
Flow velocity: 1mL/min; Detect wavelength: 250nm; Column temperature: 20 ℃; Sample size: 5 μ L.
1.2 the preparation of reference substance solution
It is an amount of to the ammonium glycyrrhizinate reference substance of constant weight that precision takes by weighing in phosphorus pentoxide desiccator drying under reduced pressure, adds dissolve with methanol, makes the solution that every 1mL contains ammonium glycyrrhizinate 0.27mg, promptly gets reference substance solution.
1.3 the preparation of need testing solution
It is 060810 pharmaceutical composition mixture 1mL of the present invention that precision is got lot number, put in the measuring bottle of 10mL, add water to scale, shake up, the accurate 1mL that draws, put in the measuring bottle of 2mL, add methanol, shake up to scale, the centrifugal 5min of 3000r/min, get supernatant, promptly get need testing solution, the preceding membrane filtration mistake of sample introduction with 0.45 μ m.
1.4 negative control experiment
Get in the negative sample of the scarce Radix Glycyrrhizae of prescription ratio and preparation technology preparation with by the preparation method of need testing solution and make negative control solution.Analyze by above-mentioned chromatographic condition sample introduction.The result shows: other compositions are noiseless to the mensuration of glycyrrhizic acid, and the HPLC spectrogram is seen Figure 1A, B, C.
1.5 linear relationship is investigated
Precision takes by weighing ammonium glycyrrhizinate reference substance 6.6mg, add methanol and make the solution that every 1mL contains ammonium glycyrrhizinate 0.66mg, accurate respectively absorption reference substance solution 0.1,0.2,0.5,1,2,5mL put in the 10mL measuring bottle, add the reference substance solution that methanol is diluted to variable concentrations, difference sample introduction 5 μ L, with the sample introduction analysis respectively of aforementioned chromatographic condition, measure its peak area concentration is carried out linear regression, it is as follows to get regression equation: Y=4248.6x-2.0685 (r=0.9998).The result shows: glycyrrhizic acid is good linear relationship with chromatographic peak area in 0.033～0.135mg/mL scope.
1.6 precision test
Withinday precision: getting concentration is the ammonium glycyrrhizinate reference substance solution of 0.033mg/mL, the accurate 5 μ L that draw, and interior on the same day sample introduction 5 times, peak area RSD is 0.77%.
Day to day precision: above-mentioned reference substance solution, in 6 days, repeat sample introduction, gained peak area RSD is 1.71%.
1.7 repeated experiment
Get 5 parts of the test samples of lot number 060830, prepare need testing solution by aforementioned need testing solution preparation method, according to 5 parts of content of aforementioned liquid phase chromatogram condition parallel assay, RSD is 0.20% (n=5) as a result, the illustration method favorable reproducibility.
1.8 average recovery experiment
The accurate pharmaceutical composition mixture of the present invention (lot number: 060830) 1mL of drawing known content, put in the measuring bottle of 10mL, totally 5 parts, accurate respectively ammonium glycyrrhizinate reference substance solution (concentration 0.660mg/mL) 1mL that adds, the preparation method preparation of pressing need testing solution then, measure, calculate average recovery, average recovery rate is 100.2%, and RSD is 0.54% (n=5), meet content assaying method and learn requirement, concrete outcome sees Table 1.
Table 1 glycyrrhizic acid average recovery result of the test (n=5)
1.9 stability experiment
Get 1.3 following need testing solutions, by above-mentioned liquid phase chromatogram condition, respectively at 0,2,4,8, the 24h sample introduction measures.Found that its peak area RSD is 5.65%, show that need testing solution is stable in 24h.
1.10 sample determination result
Get 5 batches of test sample (lot numbers 060810,060814,060830,070531,070724), be equipped with need testing solution and reference substance solution by 1.3 below legal systems, accurate respectively reference substance solution and each 5 μ L of need testing solution of drawing inject chromatograph of liquid respectively, the record peak area value, calculate the wherein content of glycyrrhizic acid, the results are shown in Table 3.
2. content Determination of Icariin is measured
2.1 chromatographic condition
Chromatographic column: Zorbax Eclipse XDB-C18 post (5 μ m, 4.6mm * 150mm);
Mobile phase: methanol: 0.01mol/L hydrochloric acid solution (55: 45);
Flow velocity: 1mL/min; Detect wavelength: 270nm; Column temperature: 20 ℃; Sample size: 5 μ L.
2.2 the preparation of reference substance solution
It is an amount of to the icariin reference substance of constant weight that precision takes by weighing in phosphorus pentoxide desiccator drying under reduced pressure, adds dissolve with methanol, makes the solution that every 1mL contains icariin 0.15mg, promptly gets reference substance solution.
2.3 the preparation of need testing solution
Precision is got this product 1mL, puts in the measuring bottle of 10mL, adds water to scale, shakes up, and the accurate 1mL that draws puts in the measuring bottle of 2mL, adds methanol to scale, shakes up, and the centrifugal 5min of 3000r/min gets supernatant, promptly gets need testing solution, the preceding membrane filtration mistake with 0.45 μ m of sample introduction.
2.4 negative control experiment
Get negative sample, make negative control solution by the preparation method of need testing solution in the scarce Herba Epimedii of prescription ratio and preparation technology preparation.Measure under above-mentioned chromatographic condition, the result shows that other compositions are noiseless to the mensuration of icariin, and its HPLC spectrogram is seen Fig. 2 A, B, C.
2.5 linear relationship is investigated
Precision takes by weighing icariin reference substance 6mg, add methanol and make the solution that every 1mL contains icariin 0.6mg, accurate respectively absorption reference substance solution 0.1,0.2,0.5,1,2,5mL put in the 10mL measuring bottle, add the reference substance solution that methanol is diluted to variable concentrations, difference sample introduction 5 μ L, distinguish the sample introduction analysis with the chromatographic condition under 2.1, measure its peak area concentration is carried out linear regression, get regression equation: y=7791.3x+17.077 (r=0.9999).The result shows: icariin is good linear relationship with chromatographic peak area in 0.015～0.15mg/mL scope.
2.6 precision test
Withinday precision: getting concentration is the icariin reference substance solution of 0.03mg/mL, the accurate 5 μ L that draw, and interior on the same day sample introduction 5 times, peak area RSD is 1.70%.
Day to day precision: above-mentioned reference substance solution, in 6 days, repeat sample introduction, gained peak area RSD is 1.99%.
2.7 repeated experiment
Get 5 parts of the test samples of lot number 060830, prepare need testing solution by the need testing solution preparation method under 2.3, according to 5 parts of content of its following liquid phase chromatogram condition parallel assay, RSD is 0.02% (n=5) as a result, illustrates that this method is easy, favorable reproducibility.
2.8 average recovery experiment
Accurate pharmaceutical composition mixture of the present invention (lot number 060830) 1mL that draws known content, put in the measuring bottle of 10mL, totally 5 parts, accurate respectively icariin reference substance solution (concentration the is 0.6mg/mL) 1mL that adds, the preparation method preparation of pressing need testing solution then, measure, calculate average recovery, average recovery rate is 100.30%, and RSD is 0.96% (n=5), meet content assaying method and learn requirement, it the results are shown in Table 2.
Table 2 icariin average recovery result of the test (n=5)
2.9 stability experiment
Get 2.3 following need testing solutions, by above-mentioned liquid phase chromatogram condition, respectively at 0,2,4,8, the 24h sample introduction measures.As a result, its peak area RSD is 2.11%, shows that need testing solution is stable in 24h.
2.10 sample determination result
Get 5 batches of test sample (lot numbers 060810,060814,060830,070531,070724), be equipped with need testing solution and reference substance solution by 2.3 below legal systems, accurate respectively reference substance solution and each 5 μ L of need testing solution of drawing inject chromatograph of liquid respectively, the record peak area value, calculate wherein content Determination of Icariin, the results are shown in Table 3.
Table 3 glycyrrhizic acid and icariin assay result
3. the assay of asperosaponin VI
3.1 reference substance solution preparation
It is an amount of to the asperosaponin VI reference substance of constant weight that precision takes by weighing in phosphorus pentoxide desiccator drying under reduced pressure, makes the reference substance solution of respective concentration with mobile phase.Microporous filter membrane filters before each the mensuration.
3.2 sample solution preparation
Precision is got pharmaceutical composition mixture of the present invention (lot number 060810) 1mL, put in the measuring bottle of 10mL, add water to scale, shake up, the accurate 1mL that draws, put in the measuring bottle of 2mL, add methanol, shake up to scale, centrifugal (3000r/min) 5min, get supernatant, promptly get sample solution, the preceding membrane filtration mistake of sample introduction with 0.45 μ m.
Negative control sample: in the prescription ratio, remove the composition of Radix Dipsaci medical material, make negative sample solution by identical preparation method.
3.3 chromatographic condition
Chromatographic column: Zorbax EcLipse XDB-C18 post (5 μ m, 4.6mm * 150mm);
Mobile phase: methanol: 0.01mol/L hydrochloric acid solution (69: 31); Flow velocity: 1mL/min;
Detect wavelength: 212nm; Column temperature: 20 ℃; Sample size: 5 μ L.
Chromatograph figure is as a result seen Fig. 3 A, B, C.
3.4 standard curve preparation
Precision takes by weighing asperosaponin VI reference substance 2.3mg, add methanol and make the solution that every 1mL contains asperosaponin VI2.3mg, accurate respectively absorption reference substance solution 0.1,0.2,0.5,1,2,5mL put in the measuring bottle of 10mL, add the reference substance solution that methanol is diluted to variable concentrations, difference sample introduction 5 μ L, distinguish the sample introduction analysis with the chromatographic condition under 3.1, measure its peak area concentration is carried out linear regression, get regression equation: y=1629.9x+72.344 (r=0.9989).The result shows: asperosaponin VI is good linear relationship with chromatographic peak area in 0.022～0.23mg/mL scope.
3.5 sample solution concentration determination
Get 5 batches of test sample (lot numbers 060810,060814,060830,070531,070724), be equipped with need testing solution and reference substance solution by 3.1 below legal systems, accurate respectively reference substance solution and each 5 μ L of need testing solution of drawing inject chromatograph of liquid respectively, the record peak area value, calculate content wherein, the results are shown in Table 4.
The assay result of table 4 pharmaceutical composition mixture of the present invention asperosaponin VI
3.6 the response rate is investigated
The accurate pharmaceutical composition mixture of the present invention (lot number: 060830) 1mL of drawing known content, put in the measuring bottle of 10mL, totally 5 parts, accurate respectively asperosaponin VI reference substance solution (concentration 0.66mg/mL) 1mL that adds, press the preparation method preparation of need testing solution then, measure, calculate average recovery.The result shows: average recovery rate 100.18%, RSD are 0.54% (n=5), meet content assaying method and learn requirement, and concrete numerical value sees Table 5.
The average recovery result of the test (n=5) of table 5 pharmaceutical composition mixture of the present invention asperosaponin VI
3.7 precision is investigated
Withinday precision: getting concentration is the asperosaponin VI reference substance solution of 2.3mg/mL, the accurate 5 μ L that draw, and interior on the same day sample introduction 5 times, peak area RSD is 1.7%.
Day to day precision: above-mentioned reference substance solution, in 6 days, repeat sample introduction, gained peak area RSD is 1.24%.
3.8 repeatability test
Get 5 parts of the test samples of lot number 060830, prepare need testing solution by the need testing solution preparation method under 3.2, according to 5 parts of content of its following liquid phase chromatogram condition parallel assay, RSD is 1.67% (n=5) as a result, the illustration method favorable reproducibility.
3.9 stability test
Get 3.2 following need testing solutions, by above-mentioned liquid phase chromatogram condition, respectively at 0,2,4,8, the 24h sample introduction measures.As a result, its peak area RSD is 5.5%, shows that need testing solution is stable in 24h.
4. content of total flavone is measured
4.1 the preparation of reference substance solution
Precision takes by weighing the icariin reference substance 4.8mg that is dried to constant weight, puts in the 20mL measuring bottle, and it is an amount of to add methanol, and ultrasonic making it dissolved, and is diluted to scale with methanol, makes the reference substance solution that every 1mL contains the 0.24mg icariin.
4.2 the preparation of need testing solution
When detecting, pharmaceutical composition mixture of the present invention dilutes 200 times, as need testing solution with methanol.
4.3 the preparation of negative sample solution
In the prescription ratio, remove the epimedium herb composition, make negative sample solution by the preparation method identical with need testing solution.
4.4 detection wavelength determination
Precision is measured above-mentioned icariin reference substance solution, need testing solution and each 2mL of negative control solution, put respectively in the 25mL measuring bottle, and the accurate respectively 0.1mol/L aluminum chloride test solution 1.0mL that adds, be diluted to scale with methanol respectively, shake up, put in 70 ℃ of water-baths heating 10min and make abundant colour developing, place 15min.With reagent is blank, according to spectrophotography, scans in 200～600nm wave-length coverage.Found that: reference substance solution and test sample all have good absworption peak at 415nm wavelength place, and negative controls is noiseless at this wavelength place, so select 415nm wavelength place to measure content of total flavone.
4.5 linear relationship is investigated
The accurate respectively reference substance solution 0mL that draws, 2.0mL, 4.0mL, 6.0mL, 6.25mL, 10mL adds to scale with methanol in the 50mL measuring bottle, shake up.Precision is measured 10.0mL respectively, respectively adds people 0.1mol/L aluminum trichloride solution 1.0mL, shakes up, heating 10min makes abundant colour developing in 70 ℃ of waters bath with thermostatic control, and take out the back room temperature and place 15min, be blank with the 1st part of sample, according to spectrophotography, measure absorbance at 415nm wavelength place.With the icariin mass concentration is abscissa, and absorbance is a vertical coordinate, the drawing standard curve, and the line linearity of going forward side by side returns, and gets regression equation: A=0.0698C+0.0008, r=0.9985.The result shows: icariin is good linear relationship in 0.096～0.48mg/ml scope.
4.6 average recovery
Precision is measured the test agent that supplies of 5 parts of known total flavones amounts, and each adds a certain amount of standard solution, and the dilution back is measured, and determination of total flavonoids is by carrying out calculate recovery rate under " linear relationship investigation " item.The results are shown in Table 6.
The average recovery of table 6 total flavones
4.7 determining of developer consumption
Precision is measured icariin reference substance solution and each 2mL of need testing solution, put respectively in the 10mL measuring bottle, each accurate aluminum chloride test solution 0.5,1.0,2.0,3.0,4.0,5.0mL that adds 0.1mol/L, be diluted to scale with methanol, shake up, heating 10min makes abundant colour developing in 70 ℃ of waters bath with thermostatic control, take out the back room temperature and place 15min, with reagent is blank, according to spectrophotography, scans in 200～600nm wave-length coverage.Found that: when aluminum chloride test solution consumption was 1ml, icariin reference substance solution and need testing solution all had good absworption peak at 415nm wavelength place.
4.8 color stability is investigated
After icariin reference substance solution and the need testing solution colour developing, place different its traps of timing.The result shows: color stability in icariin reference substance solution and the need testing solution colour developing back 15～40min, trap changes little.
4.9 repeatability test
Get same sample repeated sampling and measure 5 times, general flavone content is calculated in operation as stated above, calculates the repeatability of assay method, and the RSD meansigma methods of 5 measurement results is 0.97%.
4.10 precision test
Press method under the sample determination item, to same reference substance solution METHOD FOR CONTINUOUS DETERMINATION 5 times, record the trap value, calculating relative standard deviation RSD is 0.46%, and the result shows that this method precision is good.
4.11 sample determination
Precision is measured need testing solution 10mL during detection, and the accurate aluminum chloride test solution 1mL that adds 0.1mol/L shakes up, placing 10min in 70 ℃ of waters bath with thermostatic control, take out the back room temperature and place 15min, is blank with reagent, check by the colorimetry regulation, measure absorbance at 415nm wavelength place.According to regression equation calculation, total flavones the results are shown in Table 7 by icariin.
The measurement result of general flavone content in the table 7 pharmaceutical composition mixture of the present invention
5. the assay of total saponins
5.1 reference substance solution
Precision takes by weighing the asperosaponin VI reference substance 3.4mg that is dried to constant weight, puts in the 10mL measuring bottle, and it is an amount of to add chloroform, and ultrasonic making it dissolved, and is diluted to scale with chloroform, makes 0.34mg/mL asperosaponin VI reference substance solution.
5.2 need testing solution preparation
Precision is measured pharmaceutical composition mixture 4mL of the present invention in round-bottomed flask, adds 2mol/L hydrochloric acid 80mL in 70 ℃ of reflux 3h, with chloroform divide three times (30ml, 20ml, 10ml) reflux extraction, each 20min collects chloroform layer, is recycled to dried.Be settled in the 50ml volumetric flask with chloroform, promptly get test sample liquid.
5.3 linear relationship
The accurate asperosaponin VI standard solution 0.0mL that draws, 0.1mL, 0.2mL, 0.5mL, 1mL, 2mL, 3mL in 10mL scale test tube, water bath method.Add the vanillin-glacial acetic acid of 0.2mL 5% and the perchloric acid of 0.8mL, vortex, in 70 ℃ of water bath with thermostatic control heating 20min, take out, with the frozen water cooling, add glacial acetic acid 9mL immediately, shake up, 550nm measures down absorbance, is abscissa with asperosaponin VI mass concentration, and absorbance is that the vertical coordinate drawing standard curve line linearity of going forward side by side returns.Get linear equation: A=1629.9C+72.344, r=0.9989; The range of linearity: 0.0034～0.102mg/ml.
5.4 recovery test
Precision is measured the sample of 5 parts of known total saponin contents, each adds a certain amount of standard solution, the dilution back is measured, total saponin content is measured by measuring for test agent preparation method and condition determination, calculate recovery rate, average recovery rate is 100.84% as a result, and RSD=1.29% (n=5) the results are shown in Table 8.
Table 8 recovery test result
5.5 precision test
The above-mentioned reference substance solution of accurate absorption is measured absorbance as stated above at 550nm wavelength place, replication 5 times, and the RSD value is 1.45%, shows that precision is good.
5.6 stability test
The same need testing solution of accurate absorption, respectively at 0,2,4,8,12,24h measures, and measuring absorbance RSD is 1.25%, shows sample solution temperature in 24h.
5.7 replica test
Get same batch 5 parts in pharmaceutical composition mixture of the present invention, according to 5 parts of operation preparation sample liquid under the need testing solution preparation, press sample size algoscopy mensuration absorbance respectively, RSD is 1.16% as a result, shows that this method repeatability is good.
5.8 the mensuration of sample
The above-mentioned need testing solution 0.05mL of accurate absorption places 10mL scale test tube, and water bath method adds the vanillin-glacial acetic acid of 0.2mL 5% and the perchloric acid of 0.8mL, vortex in 70 ℃ of water bath with thermostatic control heating 20min, takes out, cool off with frozen water immediately, adding glacial acetic acid 9mL, shake up, is blank with reagent, check that by the colorimetry regulation 550nm measures the trap value down and gets, according to regression equation calculation, in asperosaponin VI, total saponins the results are shown in Table 9.
The assay result of total saponins in the table 9 pharmaceutical composition mixture of the present invention
The pharmacodynamic experiment of described pharmaceutical composition mixture is specific as follows:
One, zoopery
1.1 laboratory animal
The female sexual maturity of regular grade is 50 of the SD rats of copulation not, Mus 60～80d in age, and body weight 200 ± 20g is provided by Yueyang hospital Experimental Animal Center.Give standard periodicity of illumination (14h illumination, 10h night), indoor temperature (20 ± 2) ℃, humidity 40%～50%, standard feed and water.Raise Experimental Animal Center in Yueyang hospital.
1.2 medicine
Naltrexone is produced by Fourth Ring, Beijing pharmaceutical factory, specification 5mg/ grain; Pharmaceutical composition mixture of the present invention is provided by the Drug Manufacturing Room of the court Pharmacy department.
1.3 detection kit and instrument
Beta-endorphin (β-EP), estradiol (E
2), progesterone (P) puts inspection-free survey medicine box (The 2nd Army Medical College provides); 80-1 type centrifuge (Shanghai Surgical Operation Equipment Factory); The XW-80A eddy mixer; The DL-8R refrigerated centrifuger; SN-695B type intelligence is put and is exempted from the γ measuring instrument.
1.4 modeling method
1.4.1 screen qualified rat
All female rats are carried out vaginal smear examination every day, select the rat of oestrous cycle at 4～5d, are divided into model group and sham operated rats at random.In the rutting period of the 3rd oestrous cycle, rat is carried out autografting operation modeling and sham-operation.
1.4.2 the foundation of rat endometrium dystopy disease model (EMT)
The SD female rats, with estradiol benzoate 30 μ g/kg subcutaneous injections, behind the successive administration 3d, hydrochloric acid chlore-ammonia ketone 40mg/kg with 1%, intraperitoneal injection of anesthesia, operation is cut open the belly under 28～30 ℃ of aseptic conditions of room temperature, otch is about 1.5cm, cut a cross-talk palace at place, angle, palace, right side, be about 1cm, be placed on rapidly in the normal saline, vertically cut endometrium is separated with the flesh layer, and internal film tissue's piece of the about 5mm * 5mm of clip, lining endothelium upwards facing to body wall, is sewn in stomach wall rich blood vessel place, right side with four jiaos of piece of tissue with 7～0 cotton synthetic fibre monofilament lines.The capable end-to-end anastomosis of right side cornua uteri.Give gentamycin normal saline solution flushing abdominal cavity before closing abdomen, sew up each layer tissue at last, the regular grade environment is raised.4 weeks of postoperative, the 2nd observation of cutting open the belly, electronic caliper is measured dystopy endometrium volume, is not less than 20 cubic millimeters with volume, and the visible transparent hydrops of interior membrane vesicle is a modeling success standard.
1.4.3 sham operated rats processing method
Surgical condition and anesthesia, open the abdomen method all with the EMT model group, open behind the abdomen right side cornua uteri ligation, purpose makes sham operated rats reach the reproduction environment that is silver coin palace, one-sided palace with the EMT group.Equally with gentamycin normal saline flushing abdominal cavity, each layer tissue of layer-by-layer suture.
1.4.5 animal grouping and medication
The all same environment of all laboratory animals is raised.
Get 40 of modeling success rats, be divided into model control group (EMT group), naltrexone group, give birth unicorn mixture group, Herba Epimedii group at random, each 10, other establishes 10 of sham operated rats rats.Each treated animal is healed the wounds after the one and a half months all laboratory animals all in 8 perfusions in morning every day, naltrexone group 2mg/kgd, and the unicorn mixture group of giving birth 11g/kgd, Herba Epimedii group 2.7g/kgd, model control group and sham operated rats gavage normal saline 7.5ml/kgd.All animal successive administrations 1 month are taken a blood sample behind last 1 administration 3h, add heparin and aprotinin, detect every index.
1.5 detect index and method
The eyeball posterior vein is got blood, and radioimmunology detects rat plasma β-EP level, serum E
2With the P level.
Experimental procedure: β-each 25.6ng of EP standard substance is diluted to buffer during use that per 100 μ L contain 2.5,5,10,20,40,80,160,320,640 respectively, the standard substance of 1280pg.Concrete numerical value sees Table 10.
Table 10 detects index
E
2Identical with the detection method of P with the detection method of β-EP.
1.6 statistical method
Total data adopts the EXCEL analytic statistics, adopts one factor analysis of variance.
1.7 result
Each organizes rat proestrus β-EP horizontal detection result respectively shown in table 11,12,13.
Table 11 respectively organize rat plasma β-EP level relatively (n=10,
)
Each is organized data and compares with model group
*P＜0.01.
Table 11 result shows: model group blood plasma β-EP level raises, and has compared significant difference (P＜0.01) with sham operated rats; Naltrexone, the unicorn mixture group of giving birth β-EP level reduce, and comparing with model group all has significant difference (P＜0.01).Illustrate that thus thereby the unicorn mixture of giving birth can reach therapeutical effect by influencing β-EP level adjusting hypothalamic pituitary gonadal axis.
Table 12 is respectively organized rat blood serum E
2The level comparison (n=10,
)
Each is organized data and compares with model group
*P＜0.05.
Table 12 result shows: model group blood plasma E
2Level raises, and has compared significant difference (P＜0.05) with sham operated rats; Naltrexone, the unicorn mixture group of giving birth E
2Level reduces, and comparing with model group all has significant difference (P＜0.05).Originally experimental results show that: the unicorn mixture of giving birth can improve high E
2State reduces the growth rate of Ectopic Endometrium, and angiogenesis reduces, and blood makes the Ectopic Endometrium atrophy for reducing, and reaches thus to improve environment in the gynecopathy patient pelvic cavity, suppresses the growth of Ectopic Endometrium tissue, improves the uterus environment, helps the woman and becomes pregnant.
Table 13 respectively organize rat blood serum P level relatively (n=10,
)
Each is organized data and compares with model group
*P＜0.05.
Table 13 result shows: model group blood plasma P level raises, and has compared significant difference (P＜0.05) with sham operated rats; Naltrexone, the unicorn mixture group of giving birth P level reduce, and comparing with model group all has significant difference (P＜0.05).Originally experimental results show that: the unicorn mixture of giving birth reaches the effect that suppresses the Ectopic Endometrium growth by reducing progesterone level.
Two, cell experiment
1. material
1.1 trial drug
This pharmaceutical composition is used water saturated n-butanol extraction after making mixture, and extracting solution is concentrated into dried, is made into 2 * 10 with the culture medium that contains calf serum
-4, 2 * 10
-5, 2 * 10
-6G/ml, standby.
1.2 reagent
The DMEM/F12 culture medium, U.S. GIBCO company.Calf serum (FCS), the Hangzhou four seasons factory clearly that purifies the blood.Tetrazolium bromide (MTT), Ultra pure grade, the Amresco packing, the happy one-tenth of Easy Biotech is biological.Trypan blue (Trypan blue): Chroma import packing, Shanghai chemical reagent factory.Dimethyl sulfoxine (DMSO), AR, the Shanghai chemical reagent company limited of anticipating for a long time.D-hank ' s liquid is the reference book preparation of laboratory according to cell culture.
1.3 animal
The SD rat, female, 220～280g, Shanghai University of Chinese Medicine Affiliated Yueyang Integrated Traditional and Western Medicine Hospital zoopery center.
1.4 instrument
High speed refrigerated centrifuge RS-20III, NSK; BUCH (B-490) Rotary Evaporators, Switzerland; PH/Mv/ ℃ of meter, Cole Parmaer, Singapore; Spectra MAX 190: U.S. Molecular Devices company; NAPCO CO2 INCUBATOR:Precision Scientific; Inverted biological microscope: XSZ-D2, the optical instrument factory, Chongqing.
1.5 method
4 rats take off neck puts to death, and puts into the glass jar that fills 75% ethanol, soaks 2min.Under half aseptic state, open the abdominal cavity, ovary and around the fatty tissue of adhering to take out in the lump, put into the culture dish of sterilization rapidly, in super-clean bench, use D-Hank ' s to wash then ovary is washed several times one by one; It is residual etc. to remove unnecessary fat, fallopian tube, blood subsequently, punctures follicle at microscopically with injection needle, gently extruding, 400 order steel sieve filters, the centrifugal 5min of 800rpm, counting, and add up cell viability with trypan blue, do not see under the mirror and dye blue dead cell, so the cell motility rate is 100%; At last with 10
6Individual/mL kind plate, each concentration 6 multiple holes, the culture medium of the 4th day that more renew, pre-temperature, and to add female rats serum final concentration be 10%, makes this mixture form three different final concentrations: 10
-4, 10
-5, 10
-6(indicating with Y I, II, III respectively).Continue to cultivate, measure the cell growing state with mtt assay behind the 48h.
2. result
Table 14 is the influence data of mixture to the growth of In vitro culture rat ovary granular cell.The result shows, compares with the blank group, shows all that under three final concentrations of mixture the growth to the rat ovary granular cell has obvious effect, illustrate that pharmaceutical composition grows to gonad granulocyte facilitation is arranged, and plays a role thereby the woman become pregnant.
Table 14 mixture is to the influence of In vitro culture rat ovary granular cell growth
Three, clinical research data
1. clinical data
1.1 case source
All patients all come self-gate to examine the patient.60 routine patients are divided at random: 1. 30 examples are organized in treatment, age 24-38 year, average 31.23 ± 6.89 years old; Course of disease 3-8, average 5.56 ± 2.32 years; 2. matched group 30 examples, age 22-38 year, average 30.35 ± 7.84 years old; Course of disease 2-7, average 4.96 ± 2.85 years.Two groups of patient ages distribute, there are no significant for course of disease comparing difference, has comparability.
1.2 case is included standard in
Meet the diagnostic criteria of endometriosis and infertility,, meet qi depression to blood stasis companion's renal deficiency type and the normospermic patient of husband according to the clinical research guideline Chinese medical discrimination standard of new Chinese medicine treatment endometriosis of pelvis.
1.3 exclusion standard
Merge hysteromyoma, pelvic inflammatory disease, anogenital cancer, other part or general malignant tumor patient; Accept other therapist simultaneously; Once used Western medicine and other treatment by Chinese herbs endometriosis infertile, not enough 3 months persons of drug withdrawal; Menstrual cycle surpasses 40 days or is less than 25 days; Intentionally, vitals disease patients such as liver, kidney.
2 Therapeutic Method
2.1 treatment group
Adopt us to make the mixture that pharmaceutical composition is made by oneself: Herba Epimedii 30g, Rhizoma Polygonati 24g, Radix Dipsaci 24g, Rhizoma Cyperi 18g, Rhizoma Acori Graminei 18g, Radix Glycyrrhizae 12g.Take at twice 1 bottle of every day, 2 months is 1 course of treatment.
2.2 matched group
Adopt nemestran (the excellent Ke Fu of Britain Russell company produces, every 2.5mg) oral in menstruation beginning in the 2nd day, 2 weekly, logotype February.
2.3 observation index
2.3.1 follicular diameter is measured
Two groups of patients all go the vagina ultrasound diagnosis with monitoring follicular development situation, adopt Japanese ALoKAssD1700 colorful Doppler ultrasound diagnostic apparatus, transvaginal probe frequency 5.0MHz.Began to monitor follicular development on the 8th day from menstrual cycle, maximum follicular diameter＜10mm surveyed 1 time in per 3 days; Maximum follicular diameter 10～15mm surveyed 1 time in per 2 days; Maximum follicular diameter＞15mm person is surveyed 1 every day; Diameter＞18mm is a mature follicle.Two groups all begin monitoring in drug withdrawal after 3 months.
2.3.2 anti-endometrial antibodies (EMAb) is measured
Before the treatment and the drug withdrawal of treatment back after 3 months respectively blood sampling survey EMAb and tire.
2.3.3 tumor necrosis factor (TNF-a), interleukin-6 (IL-6) are measured
Two groups all before treatment and drug withdrawal after 3 months respectively blood sampling mensuration TNF-a, IL-6 tire.
3. observation of curative effect
3.1 clinical efficacy criterion
With reference to the curative effect determinate standard in " endometriosis combination of Chinese and Western medicine diagnosis and treatment standard ".
Recovery from illness: symptom all disappears, and local sign such as pelvic lump disappears substantially, gestation or fertility in the infertile patient 3 years.
Produce effects: symptom disappears substantially, and pelvic lump dwindles.
Effectively: sx, pelvic lump do not have and increase or slightly dwindle, and symptom does not increase the weight of in the drug withdrawal 3 months.
Invalid: cardinal symptom no change or deterioration, local patholoic change has the trend of increasing the weight of.
3.2 therapeutic outcome
The result is as shown in Table 15.
Table 15 liang group clinical efficacy relatively
Compare P＞0.05 with matched group.
3.3 two groups of maximum follicular diameter, mature follicle number averages compare
The result is shown in table 16.
The table 16 liang maximum follicular diameter of group, mature follicle number average are relatively
*Compare P＜0.05 before and after the group internal therapy.
3.4 serum EMAb potency ratio before and after two groups of treatments
The result is shown in table 17.
Serum EMAb potency ratio before and after the table 17 liang group treatment
*Compare P＜0.05 before and after the group internal therapy.
3.5 IL-6, TNF-a potency ratio are before and after two groups of treatments
The result is shown in table 18.
IL-6, TNF-a potency ratio are before and after the table 18 liang group treatment
4. discuss
Endometriosis is the women of child-bearing age's a commonly encountered diseases, and its sickness rate obviously rises in recent years, and the incidence rate of childbearing age women gynecopathy is about 5%～10%, and 80% patient has tangible dysmenorrhea, and 50% merging is sterile, has a strong impact on patient's quality of life.Endometriosis causes that infertile mechanism do not illustrate so far fully.Think that at present gynecopathy is multifactor, too many levels effect to infertile influence.Gynecopathy may cause infertility because of multiple reason, and impaired as the follicle generation, especially in middle severe gynecopathy, low-quality oocyte can reduce rate of fertilization, finally reduces embryo quality.It is generally acknowledged that blood stasis is the core pathogenesis of endometriosis.The 3rd academic conference of nineteen ninety CAIM (Chinese Association Of Integrative Medicine) department of obstetrics and gynecology Professional Committee is Blood stasis with endometriosis tcm diagnosis standard revision.On the basis of blood stasis, differences such as qi depression to blood stasis, blood stasis due to qi deficiency, blood stasis due to renal deficiency, cold blood stasis, phlegm and blood stasis, dampness and heat stasis are arranged again.The record of ametria endometrium ectopia name of disease in the Chinese medicine ancient books and records surely belongs to categories such as motherland's medical science dysmenorrhea, infertile, menoxenia, lump in the abdomen according to its clinical manifestation.Blood-stasis internal-depression has become the pathogenesis that primary disease is generally acknowledged, we are in secular practice, long according to its course of disease, the touching difficulty of the state of an illness more, the characteristics that relapse rate is high, and combination is domestic and international, and proposition vigour is lost in recent years to the result of study of primary disease pathogenic factor, and the hot blood stasis impatency of expectorant collaterals of the uterus is the pathologic basis of endometriosis.Marrow is filled up with the Rhizoma Polygonati invigorating the spleen and replenishing QI by full side, and Herba Epimedii is mended the gate of vitality, helped kidney yang is main, and it is monarch drug altogether that two medicines cooperate nourishing YIN Yiyang; Be equipped with Radix Dipsaci and strengthen the ministerial drug that act as of liver and kidney tonifying; Assistant is with the regulating menstruation of Rhizoma Cyperi (processed) depressed liver-energy dispersing and QI regulating, the comfortable air to open key of Rhizoma Acori Graminei; Radix Glycyrrhizae is for making coordinating the actions of various ingredients in a prescription.It is smart that this feasible unicorn mixture of giving birth has good the kidney warming benefit, the effect of regulating menstruation seed.Result of study shows that this mixture can obviously be alleviated gynecopathy patient symptom, and its effective percentage reaches 70%, compares the clinical efficacy no difference of science of statistics with the Western medicine nemestran.This mixture treatment endometriosis can significantly increase maximum follicular diameter, reduces serum EMAb, TNF-a and IL-6 level, thereby improves pregnancy rate.And side effect is little, relative low price, one of the effective ways for the treatment of endometriosis of can yet be regarded as.
Description of drawings
Further specify the present invention below in conjunction with the drawings and specific embodiments.
Figure 1A is the HPLC spectrogram of glycyrrhizic acid reference substance.
Figure 1B is the HPLC spectrogram of glycyrrhizic acid test sample.
Fig. 1 C is the HPLC spectrogram of glycyrrhizic acid negative control product.
Fig. 2 A is the HPLC spectrogram of icariin reference substance.
Fig. 2 B is the HPLC spectrogram of icariin test sample.
Fig. 2 C is the HPLC spectrogram of icariin negative control product.
Fig. 3 A is the HPLC spectrogram of asperosaponin VI reference substance.
Fig. 3 B is the HPLC spectrogram of asperosaponin VI test sample.
Fig. 3 C is the HPLC spectrogram of asperosaponin VI negative control product.
Wherein 1 among Figure 1A, B, the C is glycyrrhizic acid, and 2 is icariin; Among Fig. 2 A, B, the C 1 is icariin; Among Fig. 3 A, B, the C 1 is asperosaponin VI.
The specific embodiment
For technological means, creation characteristic that the present invention is realized, reach purpose and effect is easy to understand, below in conjunction with concrete diagram, further set forth the present invention.
The preparation of drug composition oral liquid:
Raw material: Herba Epimedii 300g, Rhizoma Polygonati 240g, Radix Dipsaci 240g, Rhizoma Cyperi 180g, Rhizoma Acori Graminei 180g, Radix Glycyrrhizae 120g.
Preparation: Rhizoma Cyperi and Rhizoma Acori Graminei add 12 times of water gagings, distill and extract volatile oil in 6 hours, and the aqueous solution after distillation device is in addition collected; Medicinal residues add 12 times of water gagings again and decocted 1 hour, and medical filtration is standby; 4 remaining flavors decoct 3 times with 10 times of water gagings respectively, and each 2 hours, collecting decoction, filter, filtrate and above-mentioned medicinal liquid merge, and are evaporated to relative density 1.10-1.12 (60 ℃), left standstill 24 hours, and got supernatant and reclaim ethanol and be concentrated into relative density 1.04 (20 ℃), left standstill 48 hours, get supernatant and filter, filtrate adds above-mentioned volatile oil, tween 80 and an amount of correctives, adjusts volume to 1000mL, filter, packing, sterilization promptly gets described oral liquid.
Take: one day twice, each 50ml.
The preparation of pharmaceutical composition mixture:
Raw material: Herba Epimedii 300g, Rhizoma Polygonati 240g, Radix Dipsaci 240g, Rhizoma Cyperi 180g, Rhizoma Acori Graminei 180g, Radix Glycyrrhizae 120g.
Preparation: above Six-element, add 8 times of water gagings and 4 times of water gagings decoction secondaries respectively, each back that decocts was in 85 ℃ of insulations 20 minutes, filter, merging filtrate is evaporated to relative density and is not less than 1.06 (80 ℃), and is centrifugal, get supernatant, add sucrose 200g, sodium benzoate 3g, ethyl hydroxybenzoate 0.5g, add water and make 1000mL, mixing, fill promptly gets described mixture.
Take: one day twice, each 50ml.
Embodiment 3
The preparation of pharmaceutical composition syrup:
Raw material: Herba Epimedii 300g, Rhizoma Polygonati 240g, Radix Dipsaci 240g, Rhizoma Cyperi 180g, Rhizoma Acori Graminei 180g, Radix Glycyrrhizae 120g.
Preparation: Rhizoma Cyperi and Rhizoma Acori Graminei add 12 times of water gagings, distill and extract volatile oil in 6 hours, and the aqueous solution after distillation device is in addition collected; Volatile oil becomes clathrate with the beta cyclodextrin enclose of 6 times of amounts.Medicinal residues add 12 times of water gagings again and decocted 1 hour, and medical filtration is standby; 4 remaining flavors decoct 3 times with 10 times of water gagings respectively, each 2 hours, collecting decoction filtered, filtrate and above-mentioned medicinal liquid merge, be evaporated to relative density 1.10-1.12 (60 ℃), left standstill 24 hours, get supernatant and reclaim ethanol and be concentrated into density 1.18, other gets sucrose 650g and decocts with water, after the dissolving, filter, concentrate and make syrup, with above-mentioned concentrated solution mixing, boil, put coldly, add antiseptic, above-mentioned volatile oil, tween 80, adjust volume to 1000mL, packing, sterilization promptly gets described syrup.
Take: one day twice, each 50ml.
Embodiment 4
The preparation of pharmaceutical composition tablet:
Raw material: Herba Epimedii 300g, Rhizoma Polygonati 240g, Radix Dipsaci 240g, Rhizoma Cyperi 180g, Rhizoma Acori Graminei 180g, Radix Glycyrrhizae 120g.
Preparation: Rhizoma Cyperi and Rhizoma Acori Graminei add 12 times of water gagings, distill and extract volatile oil in 6 hours, and the aqueous solution after distillation device is in addition collected; Volatile oil becomes clathrate with the beta cyclodextrin enclose of 6 times of amounts.Medicinal residues add 12 times of water gagings again and decocted 1 hour, and medical filtration is standby; 4 remaining flavors decoct 3 times with 10 times of water gagings respectively, each 2 hours, collecting decoction filtered, filtrate and above-mentioned medicinal liquid merge, be evaporated to the medicinal liquid of relative density 1.10 (60 ℃), add ethanol and make and contain alcohol amount and reach 50%, stir, left standstill 24 hours, draw supernatant and reclaim ethanol, being condensed into relative density is the extractum of 1.33-1.35 (60 ℃), gets 1 part of extractum, add 2 times of amount sucrose, 1 times of amount dextrin and ethanol are an amount of, granulate drying, beta cyclodextrin clathrate and 1% magnesium stearate mixing with volatile oil, tabletting is made 1000 altogether, promptly gets described tablet.
Take: three times on the one, each 10.
More than show and described ultimate principle of the present invention and principal character and advantage of the present invention.The technical staff of the industry should understand; the present invention is not restricted to the described embodiments; that describes in the foregoing description and the description just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (4)
1. pharmaceutical composition that is used for the treatment of female barrenness, it is characterized in that, component by weight, described pharmaceutical composition is made up of following raw materials of effective components: 30 parts of Herba Epimedii, 24 parts of Rhizoma Polygonatis, 24 parts of Radix Dipsacis, 18 parts of Rhizoma Cyperis, 18 parts of Rhizoma Acori Graminei, 12 parts in Radix Glycyrrhizae.
2. the pharmaceutical composition of treatment female barrenness as claimed in claim 1, it is characterized in that described pharmaceutical composition is an acceptable forms on pill, granule, tablet, syrup, mixture, oral liquid, drop pill, capsule, any pharmaceutics of injection.
3. the pharmaceutical composition of treatment female barrenness as claimed in claim 2 is characterized in that, described pharmaceutical composition is one or more in mixture, syrup, granule, capsule, the tablet.
4. the pharmaceutical composition of treatment female barrenness as claimed in claim 2 is characterized in that, described pharmaceutical composition is a mixture.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293995A (en) * | 1999-11-01 | 2001-05-09 | 杜桂荣 | Medicine for treating woman's acyesis |
| CN1457818A (en) * | 2002-05-16 | 2003-11-26 | 杨文志 | Poison expelling and astheniz resisting balance pill and its preparing method |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293995A (en) * | 1999-11-01 | 2001-05-09 | 杜桂荣 | Medicine for treating woman's acyesis |
| CN1457818A (en) * | 2002-05-16 | 2003-11-26 | 杨文志 | Poison expelling and astheniz resisting balance pill and its preparing method |
Non-Patent Citations (1)
| Title |
|---|
| 王霞灵，单志群，叶燕萍，黄诚，曹大农.《补肾调冲法诱发***的临床研究》.《新中医》.2003,第35卷(第2期),第19－21页. * |
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