CN101856413B - Chinese medicinal composition for soothing liver and regulating vital energy, activating blood and dissolving stasis and quality detection method - Google Patents
Chinese medicinal composition for soothing liver and regulating vital energy, activating blood and dissolving stasis and quality detection method Download PDFInfo
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- CN101856413B CN101856413B CN2009100817632A CN200910081763A CN101856413B CN 101856413 B CN101856413 B CN 101856413B CN 2009100817632 A CN2009100817632 A CN 2009100817632A CN 200910081763 A CN200910081763 A CN 200910081763A CN 101856413 B CN101856413 B CN 101856413B
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Abstract
The invention discloses a Chinese medicinal composition for soothing liver and regulating vital energy, activating blood and dissolving stasis. The composition is prepared from tangerine leaf, root of red-rooted salvia, Chinese honeylocust spine, seed of cowherb and the like and prepared into clinically or pharmaceutically acceptable formulations. The Chinese medicinal composition has good effect of dissipating breast agglomeration; and a quality detection method for the Chinese medicinal composition preparation has the advantages of good specificity, high stability, good reproducibility and high precision, is more suitable for industrialized production, and really ensures safe, effective and reliable clinical medicaments.
Description
Technical field:
The present invention relates to a kind of Chinese medicine composition and preparation method and quality determining method, particularly a kind of dispersing stagnated hepatoqi, promoting blood circulation and removing blood stasis, the Chinese medicine composition of dissolving breast mass and preparation method and quality determining method.
Background technology:
The proliferation of mammary gland is a common gynecological disease, and the incidence of disease 30% ~ 50% has certain carcinogenic tendency, and its generation is relevant with endocrinopathy especially ovarian dysfunction, to main at present sex hormone alternative medicine or the excision of adopting of the methods of treatment of the proliferation of mammary gland.But the sex hormone bad reaction is bigger, and it is often not easy for patients to accept to perform the operation yet.Motherland's traditional medicine thinks that the proliferation of mammary gland belongs to " newborn addiction ", " mammary nodule " category, and its morbidity is with most close towards wantonly two arteries and veins relation.Present composition preparation has medicines such as the red sage root, tangerine leaf that estrogen secretion is suppressed to regulate internal system makes it balance, reaches the raising immunologic function, strengthens body function, thus the treatment mammary gland disease.
Summary of the invention:
First purpose of the present invention is to provide a kind of dispersing stagnated hepatoqi, and is promoting blood circulation and removing blood stasis, the Chinese medicine composition of dissolving breast mass; Second purpose of the present invention is to provide the preparation method of this Chinese medicinal composition preparation.The 3rd purpose of the present invention is to provide the quality determining method of this Chinese medicinal composition preparation.
The present invention seeks to realize through following technical scheme:
A kind of dispersing stagnated hepatoqi of the present invention, promoting blood circulation and removing blood stasis, the raw material of the Chinese medicinal composition preparation of dissolving breast mass consists of:
Tangerine leaf 275～550 weight portions, the red sage root 275～550 weight portions, spina gleditsiae 137.5～412.5 weight portions, the seed of cowherb 137.5～412.5 weight portions, Fructus meliae toosendan 137.5～412.5 weight portions, earthworm 137.5～412.5 weight portions.
A kind of dispersing stagnated hepatoqi of the present invention, promoting blood circulation and removing blood stasis, the raw material composition of the Chinese medicinal composition preparation of dissolving breast mass is preferably:
Tangerine leaf 412.5 weight portions, the red sage root 412.5 weight portions, spina gleditsiae 275 weight portions, the seed of cowherb 275 weight portions, Fructus meliae toosendan 275 weight portions, earthworm 275 weight portions.
A kind of dispersing stagnated hepatoqi of the present invention, promoting blood circulation and removing blood stasis, the preparation method of the Chinese medicinal composition preparation of dissolving breast mass is:
Except that earthworm, the seed of cowherb, four flavor boilings such as all the other tangerine leaves 1-4 time, each 0.5-2 hour, collecting decoction filtered, and it is 1.10～1.40 (85 ℃) that filtrating is concentrated into relative density, puts cold; Earthworm, the seed of cowherb each 0.5-3 hour, filter with 50-95% alcohol reflux 1-4 time; Merging filtrate adds in the above-mentioned concentrate, and the adjustment amount of alcohol reaches 50-95%; Stir; Leave standstill, reclaim ethanol and be condensed into thick paste, according to common process; Process clinical or pharmaceutically acceptable formulation, include but not limited to dripping pill, tablet, capsule, powder, soft capsule, honeyed bolus, pill, granule, soft extract with bee honey agent, sustained release preparation, quick releasing formulation, controlled release preparation, oral liquid, ejection preparation or external preparation.
A kind of dispersing stagnated hepatoqi of the present invention, promoting blood circulation and removing blood stasis, the preparation method of the Chinese medicinal composition granules of dissolving breast mass is preferably:
Except that earthworm, the seed of cowherb, four flavor boiling secondaries such as all the other tangerine leaves, each 1 hour, collecting decoction filtered, and it is 1.25～1.30 (85 ℃) that filtrating is concentrated into relative density, puts cold, subsequent use; Earthworm, the seed of cowherb 2 hours for the first time, 1 hour for the second time, filter with 70% alcohol reflux secondary; Merging filtrate adds in the above-mentioned concentrate, and the adjustment amount of alcohol reaches 70%, stirs; Leave standstill, reclaim ethanol and also be condensed into thick paste, an amount of with sucrose 500 weight portions and starch, dextrin, mixing; Process particle, drying is processed promptly and is got.
A kind of dispersing stagnated hepatoqi of the present invention, promoting blood circulation and removing blood stasis, the preparation method of the Chinese medicine composition tablet of dissolving breast mass is preferably:
Above Six-element, except that earthworm, the seed of cowherb, four flavor boiling secondaries such as all the other tangerine leaves, each 1 hour, collecting decoction filtered, and it is 1.25～1.30 (85 ℃) that filtrating is concentrated into relative density, puts cold, subsequent use; Earthworm, the seed of cowherb 2 hours for the first time, 1 hour for the second time, filter merging filtrate with 70% alcohol reflux secondary; Add in the above-mentioned concentrate, the adjustment amount of alcohol reaches 70%, stirs, and leaves standstill, and reclaims ethanol and is concentrated into thick paste; Be dried to dry extract, pulverize, add right amount of auxiliary materials, mixing is processed particle; Drying is pressed into 1000, and sugar coating promptly gets.
A kind of dispersing stagnated hepatoqi of the present invention, promoting blood circulation and removing blood stasis, the preparation method of the Chinese medicinal composition capsules agent of dissolving breast mass is preferably:
Above Six-element, except that earthworm, the seed of cowherb, four flavor boiling secondaries such as all the other tangerine leaves, each 1 hour, collecting decoction filtered, and it is 1.25～1.30 (85 ℃) that filtrating is concentrated into relative density, puts cold, subsequent use; Earthworm, the seed of cowherb 2 hours for the first time, 1 hour for the second time, filter with 70% alcohol reflux secondary; Merging filtrate adds in the above-mentioned concentrate, and the adjustment amount of alcohol reaches 70%, stirs; Leave standstill, reclaim ethanol and also be condensed into thick paste, an amount of with sucrose 500g and starch, dextrin, mixing; Process particle, drying, encapsulated, promptly get.
The present invention provides the method for quality control of this Chinese medicinal composition preparation, and the method comprising the steps of:
A, assay: chromatographic condition and system suitability test, use octadecylsilane chemically bonded silica to be filling agent; (30-50: 50-70) methyl alcohol of ratio-0.01～0.03mol/L phosphoric acid solution (0.01～0.03mol/L phosphoric acid solution 1000ml regulates pH to 2.5～3.5 with triethylamine) is a moving phase; The detection wavelength is 280～290nm.The preparation of reference substance solution: it is an amount of that precision takes by weighing the aurantiamarin reference substance, adds methyl alcohol and process the solution that every 1ml contains 2～10 μ g.The preparation of need testing solution: get said composition preparation porphyrize, get 0.5g, the accurate title, decide, and puts in the conical flask, adds the about 20～80ml of methyl alcohol; Sonicated 20～40 minutes is put coldly, filters, and filtrating is put in 25～100ml measuring bottle; Filter residue and container be with methanol wash 1～3 time, each 3～10ml, and washing lotion is filtered in the same measuring bottle, adds methyl alcohol to scale; Shake up, filter, get subsequent filtrate, promptly get.Determination method: accurate respectively reference substance solution and each 10～20 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly get, the composite preparation that is equivalent to crude drug 16-22g contains the tangerine leaf with aurantiamarin (C
28H
34O
15) meter, must not be less than 3.0mg;
B, discriminating: get said composition preparation 10g, porphyrize adds water 30～50ml and makes dissolving, and adding watery hydrochloric acid adjusting pH value is 3～4; Extract 1～3 time with the ethyl acetate jolting, each 20～50ml merges ethyl acetate liquid; Evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the protocatechualdehyde reference substance, adds methyl alcohol and processes the solution that every 1ml contains 1～3mg, as reference substance solution.Draw each 2～10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, so that (4-12: 4-7: 0.8) benzene-ethyl acetate of ratio-formic acid mixed solvent is a developping agent; Launch; Take out, dry, spray is with 1% liquor ferri trichloridi-1% potassium ferricyanide (1: 1).In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color.
C, discriminating: get said composition preparation 10g, porphyrize adds chloroform 10～30ml, close plug, and sonicated 15～30 minutes filters, and filtrating is concentrated into 1ml, as need testing solution.Other gets earthworm control medicinal material 1g, shines medicinal material solution in pairs with legal system, according to the thin-layered chromatography test; Draw each 2～10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, so that (13-6: 1) benzene of ratio-ethyl acetate mixed solvent is a developping agent; Launch; Take out, dry, to ultraviolet lamp (365nm), inspect.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color.
Above-mentioned method of quality control preferably includes following assay:
Assay: chromatographic condition and system suitability test, use octadecylsilane chemically bonded silica to be filling agent; Methyl alcohol-0.025mol/L phosphoric acid solution (85% phosphoric acid solution 1.70ml is diluted with water to nearly 1000ml, and about 1.8ml regulates pH to 2.9～3.1 with triethylamine, adds water to 1000ml) (40: 60) is moving phase; The detection wavelength is 284nm.Number of theoretical plate calculates by the aurantiamarin peak should be not less than 5000.The preparation of reference substance solution: it is an amount of that precision takes by weighing the aurantiamarin reference substance, adds methyl alcohol and process the solution that every 1ml contains 5 μ g, promptly gets.The preparation of need testing solution: get the said composition preparation, porphyrize is got 0.5g, and accurate the title decides, and puts in the conical flask; Add the about 40ml of methyl alcohol, sonicated (power 250W, frequency 33KHZ) 30 minutes is put coldly, filters; Filtrating is put in the 50ml measuring bottle, and filter residue and container be with methanol wash 2 times, 4ml at every turn, and washing lotion is filtered in the same measuring bottle, adds methyl alcohol to scale; Shake up, filter, get subsequent filtrate, promptly get.Determination method: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly get.
Preferably include following differential method in the above-mentioned method of quality control:
Red sage root thin layer is differentiated: get said composition preparation 10g, porphyrize adds water 40ml and makes dissolving, and adding watery hydrochloric acid adjusting pH value is 3～4; Extract secondary with the ethyl acetate jolting, each 30ml merges ethyl acetate liquid; Evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution.Other gets the protocatechualdehyde reference substance, adds methyl alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution.Drawing each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is developping agent with benzene-ethyl acetate-formic acid (8: 5: 0.8), launches, and takes out, and dries, and spray is with 1% liquor ferri trichloridi-1% potassium ferricyanide (1: 1).In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color.
The earthworm thin layer is differentiated: get said composition preparation 10g, porphyrize adds chloroform 20ml, close plug, and sonicated 20 minutes filters, and filtrating is concentrated into 1ml, and as need testing solution, other gets earthworm control medicinal material 1g, shines medicinal material solution in pairs with legal system.Drawing each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is developping agent with benzene-ethyl acetate (9: 1), launches, and takes out, and dries, and to ultraviolet lamp (365nm), inspects.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color.
Above-mentioned method of quality control Chinese medicinal composition preparation preferred feedstock consists of: the granule of tangerine leaf 412.5g, red sage root 412.5g, spina gleditsiae 275g, seed of cowherb 275g, Fructus meliae toosendan 275g, earthworm 275g, and through the preparation of following method: above Six-element, except that earthworm, the seed of cowherb; Four flavor boiling secondaries such as all the other tangerine leaves, each 1 hour, collecting decoction; Filter; It is 1.25～1.30 (85 ℃) that filtrating is concentrated into relative density, puts cold, subsequent use; Earthworm, the seed of cowherb 2 hours for the first time, 1 hour for the second time, filter with 70% alcohol reflux secondary; Merging filtrate adds in the above-mentioned concentrate, and the adjustment amount of alcohol reaches 70%, stirs; Leave standstill, reclaim ethanol and also be condensed into thick paste, an amount of with sucrose 500g and starch, dextrin, mixing; Process particle, drying promptly gets.
Chinese medicine composition of the present invention has good dispersing stagnated hepatoqi, and is promoting blood circulation and removing blood stasis, function for dissolving breast mass.
The quality determining method of Chinese medicinal composition preparation of the present invention; Warp experiment proof: content assaying method of the present invention detects good, the stable height of specificity, favorable reproducibility, precision height; Be fit to industrialized production more, really guaranteed safety of clinical administration, effective, reliable.
Following experimental example and embodiment are used to further specify but are not limited to the present invention.
The preparation that all experimental examples of the present invention are selected for use is the granule of the present invention that makes according to the embodiment of the invention 1, but experimental result of the present invention is not limited in this granule.
Experimental example 1: present composition preparation anti-experimental character rat mammary gland proliferative effect research
1. experiment material:
Medicine and reagent: present composition preparation; Estradiol benzoate injection; Progesterone injection; Hormone determination is used various radioimmunological kits.Animal: the SD rat, female, body weight 200 ~ 250g.Instrument: MOTIC biology microscope IMAQ analytic system.
2. experimental technique
10 age in week 60 of SD female rats, body weight 200-250g leaves and takes 10 at random as normal control, all the other 50 rats are duplicated hyperplasia of mammary gland model, at first intramuscular injection oestradiol benzoate 0.5mgkg
-1D
-1, once a day, 20d changes intramuscular injection progesterone 5mgkg then into continuously
-1D
-1, once a day, continuous 5d.Rat is further divided into 5 groups at random after the modeling, 10 every group, irritates the high, medium and low dosage of stomach present composition preparation respectively and (is respectively 10,5,2.5g crude drug kg
-1), normal control group and model control group are irritated stomach with the equal-volume tap water, and the administration volume is 20mLkg
-1, continuous 35d.Next day after the last administration, rat etherization, heart extracting blood, 2000rmin
-1Centrifuging serum; Put the method for exempting from and measure hypophysis prolactin (PRL), estradiol (E2), progesterone female, progesterone levels such as (P); Measure rat the 2nd, 3 pairs of height of nipples and diameters with vernier scale, and get second pair of mammary gland and be fixed in 10% formalin pathological section behind the FFPE (every side mammary gland is cut two sections in length and breadth); HE dyeing back microscopy is graded by table 1 grading standard.
Table 1 proliferation of mammary gland standards of grading
3. experimental result:
3.1 influence to proliferation of mammary gland rat height of nipples and diameter:
Rat the 2nd after the modeling; The 3 pairs of height of nipples and papilla diameter obviously increase; Compared notable difference (P＜0.05) with the normal control group, height of nipples is starkly lower than model control group (P＜0.05) after the middle and high dosage medication of present composition preparation, and papilla diameter also has reduction trend.See table 2.
Table 2 present composition preparation is to the influence of proliferation of mammary gland rat height of nipples
Annotate: compare with the normal control group,
*P＜0.05; Compare with model control group,
*P＜0.05
3.2 influence to proliferation of mammary gland rat blood serum sex hormone level:
Rat blood serum hypophysis prolactin (PRL), estradiol (E2) content are significantly higher than the normal control group after the modeling; Progesterone (P) then significantly is lower than control group; Testosterone (T) slightly reduces; Each dose groups of present composition preparation all has certain antagonism to above-mentioned change, middle and high dose groups PRL, and E2 and P content are compared with model group all has significant difference.See table 3.
3.3 influence to proliferation of mammary gland rat mammary gland Histopathology:
The mammary gland pathological section mirror down lobule of mammary gland quantity of visible rats in normal control group is few, sparse distribution, and acinus is few in the leaflet, and general 2-4, the acinus epithelium is cube, and alveolar lumen is little, no secretion or a little secretion is only arranged.The equal hyperplasia of the mammary gland of the nearly all rat of model control group: leaflet quantity increases greatly.Rats in normal control group mammary gland, body of gland quantity is few, sparse distribution, acinus is few in the leaflet; General 2 ~ 4, alveolar lumen is little, and acinus is not expanded, and the acinus epithelium is cube; No secretion or a little secretion is only arranged is in state repose period, and conduit is not expanded, and does not have secretion.
The middle and high dose groups rat mammary gland of present composition preparation leaflet significantly reduces than model group, and is only many slightly than the normal control group, and the acinus number also significantly reduces in each leaflet, does not see secretion.Concrete rating result is seen table 4, analyzes through Ridit, and the middle and high dose groups of present composition preparation has the obvious treatment effect to the rat mammary gland hyperplasia.
Experimental result shows that present composition preparation has clear and definite therapeutic action to rat experiment property hyperplasia of mammary gland model.
Experimental example 2: pharmacodynamic experiment:
The neuron of present composition preparation for treating functional dyspepsia FD and neurotransmitter experimental study.
1, experiment material:
Present composition preparation; Not husky Billy, β-NADPH, N-BT, TritonX2100, Tris damping fluid, nitrogen monoxide kit, acetylcholinesterase are measured kit.Animal: SD is a rat, body weight (200 ± 20) g, male and female half and half.Instrument: medical science color image analyser HPIAS2500 type
2, experimental technique and result:
2.1 present composition preparation is to NOS positive neuron distribution area in the rat stomach hole tissue and active influence experiment.
Animal divides into groups to get 30 rats, is divided into 3 groups at random: present composition preparation group, positive controls and blank group, 10 every group.Present composition preparation group, positive controls and blank group are irritated stomach respectively and are given present composition preparation soup, not husky Billy's soup and physiological saline.3 treated animal dosages are the 5.0ml/kg body weight, and present composition preparation adds water, and to make drug concentration be 0.486g/ml, and not husky Billy grinds into powder, add water and join and be 0.27g/L, and the blank group gives isopyknic physiological saline.Behind the animal-use drug the 14th day, behind 60min after the administration, put to death animal, get at stomach Dou Qu (apart from pylorus 0.5cm) and organize 1mm
3,, reenter 25% (4 ℃) sucrose and spend the night with the fixing 24h (4 ℃) of 4% formaldehyde.
Detection method with tissue specimen with the continuous frozen section of AO cryostat, thickness 20 μ m, NADPH2 diaphorase group method dyeing shows the NOS positive neuron, fluorescent microscope is observed down, the quantitative test of medical science color image analyser image.
Each is organized data and all adopts mean ± standard deviation to represent, carries out data processing with the SPSS statistical software, and one-way analysis of variance compares between all employing groups in twos.Experimental result is seen table 5.
Table 5 present composition preparation is to NOS positive neuron distribution area in the rat stomach hole tissue and active influence experiment
Compare with the blank group,
*P＜0.05,
*P＜0.01; Compare with positive controls,
△P＜0.05,
△ △P＜0.01; A: parameter A is represented the area of NOS positive neurons fiber in the measurement window; M: parameter M representes the AO of NOS positive neurons fiber in the measurement window; N=10
2.2 present composition preparation is to NO in the rat stomach hole tissue, Ach influences experiment
Animal divides into groups to get 30 rats, is divided into 3 groups at random: present composition preparation group, positive controls and blank group, 10 every group.Present composition preparation group, positive controls and blank group are irritated stomach respectively and are given present composition preparation soup, not husky Billy's soup and physiological saline.3 treated animal dosages are the 5.0ml/kg body weight, and present composition preparation adds water, and to make drug concentration be 0.486g/ml, and not husky Billy grinds into powder, add water and join and be 0.27g/L, and the blank group gives isopyknic physiological saline.Animal was drawn materials after the medication the 14th day, behind administration 60min, put to death animal, got at stomach Dou Qu (apart from pylorus 0.5cm) and organized 400mg, added 4ml physiological saline, homogenate, and the centrifugal 15min of 3500r/min gets supernatant and in-20 ℃ of refrigerators, preserves to be measured.
The detection method that detection method NO, Ach all provide according to corresponding reagent box instructions detects.
Each is organized data and all adopts mean ± standard deviation to represent, carries out data processing with the SPSS statistical software, and one-way analysis of variance compares between all employing groups in twos.Experimental result is seen table 6.
Table 6 present composition preparation is to NO in the rat tissue, the influence of Ach
Compare with the blank group,
*P＜0.05,
*P＜0.01; Compare with positive controls,
△P＜0.05,
△ △P＜0.01;
Experimental result shows; Preliminary observation under fluorescent microscope; Present composition preparation group rat stomach hole mucous membrane intramuscular NOS positive neurons fiber reduces, attenuates; Dyeing weakens, and sees seldom that at submucosa the positive neuron cell space reaches from inner process, and positive controls positive products and present composition preparation category are seemingly.But the blank group stomach hole mucous membrane intramuscular NOS of portion positive neuron obviously increases slightly, increases than present composition preparation group and positive controls, and dyeing strengthens.We can see from table 1; The area of present composition preparation group and positive controls stomach hole mucous membrane intramuscular NOS positive neurons fiber and tip thereof obviously reduces than the blank group; And its mean light absorbency value also obviously reduces than the blank group, has significant differences (P＜0.01).We can see from table 2, and NO content obviously reduces in present composition preparation group and the positive controls rat stomach hole tissue, and Ach content increases, and with the blank group significant differences (P＜0.01) are arranged all.
We analyze thus, the function of the short gastric emptying of present composition preparation, and with the content of excitatory neurotransmitter Ach in its increase gastric tissue, it is relevant to reduce in the gastric tissue inhibiting nerve mediator NO content simultaneously.The content of NO reduces with present composition preparation and reduces NOS positive neurons metamember in the rat stomach hole wall, and it is relevant to reduce its activity.
Experimental example 3: orange peel content assaying method experiment
Preparation compsns of the present invention is made up of 6 flavor Chinese medicines such as orange peel, the red sage root, earthworm.Aurantiamarin is the effective constituent that mainly contains of orange peel.Aurantiamarin is a kind of important flavone compound in the orange peel chemical constitution, and " Chinese pharmacopoeia version I in 2005 portion is with the reference substance of aurantiamarin as the high performance liquid chromatography assay of orange peel.Determination of Hesperidin Content helps to control the quality of this Chinese patent medicine preparation in the mensuration present composition, guarantees safety of clinical administration, effective.
The high effective liquid chromatography for measuring content of hesperidin is adopted in this experiment, and analysis speed is fast, and is highly sensitive, and specificity is strong, applied range, and reappearance and accuracy all are superior to thin-layer chromatography-ultraviolet spectrophotometry, TLCS.
1, measures the selection of wavelength
Carry out the ultraviolet full wavelength scanner with moving phase dilution back aurantiamarin reference substance solution; The result shows that its absorption maximum is at 284nm; Therefore select the mensuration wavelength of 284nm wavelength as aurantiamarin in this preparation group thing; Can effectively improve the sensitivity and the accuracy of mensuration, reduce the interference that other impurity brings.
2, the selection of chromatographic condition
2.1 the selection of chromatographic column:
The chromatographic column that to adopt three kinds of octadecylsilane chemically bonded silicas be filling agent experimentizes, and the chromatographic column model is following, and the result shows, the chromatographic column that three kinds of octadecylsilane chemically bonded silicas are filling agent all is suitable for.
Hypersil?ODS?C
18(250mm×4.6mm)；
AQ-C
18?5μm?250mm×4.6mm(AICHROM)；
YMC-ODS-3?C
18?5μm?150mm×4.6mm。
2.2 the selection of moving phase:
Select acetonitrile-water-phosphoric acid (20: 80: 0.1) and methanol-water-acetic acid (35: 61: 4) to investigate for moving phase.Experimental result shows that the aurantiamarin peak symmetry is poor than the former in latter's chromatogram, and retention time is long than the former simultaneously.
3, the preparation of need testing solution
In order to ensure measuring real result, accurate, the present invention extracts the aurantiamarin in the present composition particle fully when guaranteeing to prepare need testing solution through a series of investigations.
3.1 the selection of method for distilling
Get present composition preparation porphyrize, get 0.5g, the accurate title, decide, and puts in the conical flask, adds the about 40ml of methyl alcohol, claims to decide weight, shines following method test: a: sonicated 30 minutes; B: reflux 30 minutes; C: cable-styled extraction 60 minutes.Put coldly, filter, filtrating is put in the 50ml measuring bottle, and filter residue and container be with methanol wash 2 times, each 4ml, and washing lotion is filtered in the same measuring bottle, adds methyl alcohol to scale, shakes up, and filters, and gets subsequent filtrate, filtrates as need testing solution.Accurate each the 10 μ l of need testing solution 1-3 that draw inject liquid chromatograph, measure.Table 7 is seen in the selection of method for distilling.
Table 7
Above data show that the ultrasonic Extraction content of hesperidin is higher, and ultrasonic method is simple and easy to do, so select ultrasonic Extraction.
3.2 extraction choice of Solvent:
Get present composition preparation porphyrize, get 0.5g, the accurate title, decide, put in the conical flask, and according to following method test, the accurate a that adds: methyl alcohol; B:80% methyl alcohol; Each 40ml of c:50% methyl alcohol, sonicated 30 minutes is put coldly, filters, and filtrating is put in the 50ml measuring bottle; Filter residue and container be with using above-mentioned solvent wash 2 times respectively, each 4ml, and washing lotion is filtered in the same measuring bottle, adds methyl alcohol to scale; Shake up, filter, get subsequent filtrate, filtrating is as need testing solution.Accurate each the 10 μ l of need testing solution 1-3 that draw inject liquid chromatograph, measure.Extract choice of Solvent and see table 8.
Table 8
Above data show that methyl alcohol is for extracting the aurantiamarin optimum solvent, so select methyl alcohol as extracting solvent.
3.3 the selection of extraction time:
Get present composition preparation porphyrize, get 0.5g, the accurate title, decide, and puts in the conical flask, adds the about 40ml of methyl alcohol; According to following method test: sonicated is 15,30,45 minutes respectively, puts coldly, filters, and filtrating is put in the 50ml measuring bottle; Filter residue and container be with methanol wash 2 times, each 4ml, and washing lotion is filtered in the same measuring bottle, adds methyl alcohol to scale; Shake up, filter, get subsequent filtrate, filtrating is as need testing solution.Accurate each the 10 μ l of need testing solution 1-3 that draw inject liquid chromatograph, measure.Table 9 is seen in the selection of extraction time.
Table 9
Above data show, can aurantiamarin in the preparation be extracted fully basically in ultrasonic 30 minutes, so ultrasonic Extraction 30 minutes.
3.4 extract the preferred of quantity of solvent:
Get present composition preparation porphyrize, get 0.5g, the accurate title, decide, and puts in the conical flask, shines following method test: accurate respectively methyl alcohol 25,40, the 50ml of adding; Sonicated 30 minutes is put coldly, filters, and filtrating is put in the 50ml measuring bottle; Filter residue and container are used washing 2 times, each 4ml, and washing lotion is filtered in the same measuring bottle, adds methyl alcohol to scale; Shake up, filter, get subsequent filtrate, filtrating is as need testing solution.Accurate each the 10 μ l of need testing solution 1-3 that draw inject liquid chromatograph, measure.That extracts quantity of solvent preferably sees table 10.
Table 10
Above data show that 40ml methyl alcohol can extract the aurantiamarin in the preparation fully basically, so confirm that extracting quantity of solvent is 40ml.
Chromatographic condition that to sum up content of hesperidin is measured in the last definite present composition granule of test and reference substance and test sample preparation method are:
Chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filling agent; Methyl alcohol-0.025mol/L phosphoric acid solution (85% phosphoric acid solution 1.70ml is diluted with water to nearly 1000ml, and about 1.8ml regulates pH to 2.9～3.1 with triethylamine, adds water to 1000ml) (40: 60) is moving phase; The detection wavelength is 284nm.The preparation of reference substance solution: it is an amount of that precision takes by weighing the aurantiamarin reference substance, adds methyl alcohol and process the solution that every 1ml contains 5 μ g, promptly gets.The preparation of need testing solution: get present composition preparation porphyrize, get 0.5g, the accurate title, decide, and puts in the conical flask, adds the about 40ml of methyl alcohol; Sonicated 30 minutes is put coldly, filters, and filtrating is put in the 50ml measuring bottle; Filter residue and container be with methanol wash 2 times, each 4ml, and washing lotion is filtered in the same measuring bottle, adds methyl alcohol to scale; Shake up, filter, get subsequent filtrate, promptly get.Determination method: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly get.
Experimental example 4: content of hesperidin assay method confirmatory experiment
Detecting instrument (room temperature detection): TSP high performance liquid chromatograph;
Chromatographic column: Hypersil ODS C
18(250mm * 4.6mm);
Moving phase: acetonitrile-water-phosphoric acid (20: 80: 0.2)
Detect wavelength: 284nm
Flow velocity: 1.0mL/min
Reference substance source: aurantiamarin (available from Nat'l Pharmaceutical & Biological Products Control Institute)
Test sample lot number: 04081201,04091302,04101403
The test sample preparation: get present composition preparation porphyrize, get 0.5g, the accurate title, decide, and puts in the conical flask, adds the about 40ml of methyl alcohol; Sonicated 30 minutes is put coldly, filters, and filtrating is put in the 50ml measuring bottle; Filter residue and container be with methanol wash 2 times, each 4ml, and washing lotion is filtered in the same measuring bottle, adds methyl alcohol to scale; Shake up, filter, get subsequent filtrate, promptly get.
The preparation of reference substance solution: it is an amount of that precision takes by weighing the aurantiamarin reference substance, adds methyl alcohol and process the solution that every 1ml contains 5 μ g, promptly gets.
1. content assaying method is learned and is investigated:
1.1 linear relationship is investigated:
Accurate reference substance solution 2.5,5,10,15, the 20 μ L that draw measure aurantiamarin absorption peak area.With the integrated value of aurantiamarin absorption peak area to aurantiamarin sample size drawing standard curve, regression equation: A=2562.8X+25.7, r=0.9999.
The result shows that aurantiamarin has good linear relationship in 0.0125～0.1 μ g scope.
1.2 precision test:
The accurate reference substance solution of drawing repeats sample introduction 6 times, measures aurantiamarin absorption peak area, and RSD is 1.0% as a result.
1.3 repeated experiment:
(lot number: 04081201) sample is five parts, and every part is measured, and tries to achieve relative standard deviation to get same lot number.RSD is 1.2% as a result.
1.4 the stability test of solution:
Accurate same (04081201) need testing solution of drawing, aurantiamarin absorption peak area 6 times are measured at every interval 2 hours, and RSD is 0.4% as a result, shows that need testing solution is basicly stable in 8h.
1.5 recovery test:
Adopt the application of sample absorption method, precision takes by weighing the present composition preparation 0.25g of known content, and accurate the title decides, and the accurate aurantiamarin reference substance that adds is an amount of, presses need testing solution preparation method preparation, measures this method aurantiamarin recovery.The recovery test result sees table 11.
Table 11
1.6 measuring the result, present composition preparation sees table 12:
Table 12
According to above data, content limit is decided to be: present composition preparation contains orange peel in aurantiamarin for every bag, and every bag must not be less than 0.3mg.
Experimental example 5: red sage root thin layer discrimination test
Effective constituent in the present composition preparation in the contained red rooted salvia is aldehyde compounds such as protocatechualdehyde.The present invention utilizes thin-layer chromatography to differentiate the protocatechualdehyde in the preparation, identifies the red rooted salvia in the preparation.
1, the selection of thin layer chromatography:
Common thin-layer chromatography has silica gel thin-layer chromatography, ply of paper to analyse etc.Silica gel thin-layer is applicable to low polarity and middle polarity composition, and the scope of application is wider; Ply of paper is analysed and is applicable to high polarity composition such as polysaccharide composition.Protocatechualdehyde is a middle polarity, selects silica gel column chromatography to experimentize.
2, the preparation of test sample:
2.1 the method for distilling of protocatechualdehyde is investigated in the preparation:
Method 1: Direx process
Get present composition preparation 10g, porphyrize adds water 40ml and makes dissolving, extracts secondary with the ethyl acetate jolting, and each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution 1.
Method 2: sour water extraction
Get present composition preparation 10g, porphyrize adds water 40ml and makes dissolving, and adding watery hydrochloric acid adjusting pH value is 3～4, extracts secondary with the ethyl acetate jolting, and each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution 2.
Method 3: liquid-solid extraction method
Get present composition preparation 10g, porphyrize, with 60ml ethyl acetate ultrasonic Extraction, 30min merges ethyl acetate liquid, and evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution 3.
Get the protocatechualdehyde reference substance, add methyl alcohol and process the solution that every 1ml contains 1mg, as reference substance solution.
Drawing each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is developping agent with benzene-ethyl acetate-formic acid (8: 5: 0.8), launches, and takes out, and dries, and spray is with 1% liquor ferri trichloridi-1% potassium ferricyanide (1: 1).
Experimental result is following:
Need testing solution 1 is thickness, muddiness very, point sample difficulty, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on the spot of no same color.
On the corresponding position of protocatechualdehyde reference substance chromatogram, show the spot of same color in need testing solution 2 chromatograms, speckle displacement confirms that it is clear to develop the color.
Ingredient is more in need testing solution 3 chromatograms, on the corresponding position of protocatechualdehyde reference substance chromatogram, shows the spot of same color,, but have impurity to disturb;
Therefore, select for use the sour water extraction to extract protocatechualdehyde in the preparation.
2.2 the investigation of extracting liquid pH value:
The acidity of solution can influence the percentage extraction of protocatechualdehyde in the preparation need testing solution process.
Get three parts of present composition preparation 10g, porphyrize adds water 40ml and makes dissolving; Adding watery hydrochloric acid adjusting pH value respectively is 1～2,3～4,5～6, extracts secondary with the ethyl acetate jolting respectively, each 30ml; Merge ethyl acetate liquid; Evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution 1-3.
Get the protocatechualdehyde reference substance, add methyl alcohol and process the solution that every 1ml contains 1mg, as reference substance solution.
Drawing each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is developping agent with benzene-ethyl acetate-formic acid (8: 5: 0.8), launches, and takes out, and dries, and spray is with 1% liquor ferri trichloridi-1% potassium ferricyanide (1: 1).
Experimental result shows:
It is in 1～2 test sample chromatogram that watery hydrochloric acid is regulated pH value, with the corresponding position of reference substance chromatogram on the apparent more weak spot of color; It is in 3～4 test sample chromatograms that watery hydrochloric acid is regulated pH value, with the corresponding position of reference substance chromatogram on the spot of apparent same color; It is in 5～6 test sample chromatograms that watery hydrochloric acid is regulated the pH value, with the corresponding position of reference substance chromatogram on the spot of no same color.Almost do not play the effect that suppresses conversion.
Therefore, select for use watery hydrochloric acid that extracting liquid pH value is adjusted to 3～4.
2.3 the investigation of extracting process
Adopt L
93
(4)The orthogonal experiment design is investigated the ethyl acetate extraction method.See table 13.
Table 13
9 parts of need testing solutions have been prepared through above-mentioned experimental program.
Other gets the protocatechualdehyde reference substance, adds methyl alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution.
Drawing each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is developping agent with benzene-ethyl acetate-formic acid (8: 5: 0.8), launches, and takes out, and dries, and spray is with 1% liquor ferri trichloridi-1% potassium ferricyanide (1: 1).
Experimental result is following:
When adopting sherwood oil to make extraction solvent, extraction efficiency is lower, in the test sample chromatogram with reference substance chromatogram relevant position on, do not have corresponding spot; When adopting normal butyl alcohol to make extraction solvent, extracting power is strong excessively, and a lot of other compositions in the red sage root also are extracted out, in the test sample chromatogram, with reference substance chromatogram relevant position on the spot of apparent same color is arranged, but impurity is more, disturbs and differentiates; When adopting ethyl acetate to make extraction solvent, extracting power is suitable, relatively factors such as consumption, number of times and water consumption; Discovery adds water 40ml (4 times of amounts) makes dissolving, and adding watery hydrochloric acid adjusting pH value is 3～4, extracts 2 times with the ethyl acetate jolting; Each 30ml merges ethyl acetate liquid.By the test sample of this method preparation, with the corresponding position of reference substance chromatogram on show the spot of same color, clear, noiseless.
2.4 the investigation of need testing solution and control medicinal material solution point sample amount:
Drawing need testing solution and control medicinal material solution 2 μ l, 5 μ l, 10 μ l, put respectively on same silica gel g thin-layer plate, is developping agent with benzene-ethyl acetate-formic acid (8: 5: 0.8); Launch; Take out, dry, spray is with 1% liquor ferri trichloridi-1% potassium ferricyanide (1: 1).
Experimental result is following:
When protocatechualdehyde reference substance solution point sample amount was 2 μ l, spot intensity was lower, should not observe; When the point sample amount was 5 μ l, clear spot on the protocatechualdehyde reference substance solution thin layer was of moderate size; When the point sample amount was 10 μ l, spot was too big, hangover.
The best point sample amount of confirming the protocatechualdehyde reference substance solution is 5 μ l.
When need testing solution point sample amount was 2 μ l, spot intensity was lower on the corresponding position of protocatechualdehyde reference substance, should not observe; When the point sample amount was 5 μ l, spot was obvious on the corresponding position of protocatechualdehyde reference substance, and spot size is moderate, and front and back are noiseless; When the point sample amount was 10 μ l, spot was bigger on the corresponding position of protocatechualdehyde reference substance, and obviously hangover links to each other with the front and back spot.
The best point sample amount of confirming test sample is 5 μ l.
2.5 the selection of developping agent:
Draw each 5 μ l of need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, launch with following developping agent respectively, take out, dry, spray is with 1% liquor ferri trichloridi-1% potassium ferricyanide (1: 1).
Experimental technique and result are following:
Method 1: developping agent is that benzene-ethyl acetate-formic acid (3: 5: 0.8) is developping agent, and developping agent polarity is bigger, and the protocatechualdehyde spot does not separate with the front and back spot in the test sample chromatogram;
Method 2: developping agent is benzene-ethyl acetate-formic acid (8: 5: 0.8), and polarity is suitable, and the protocatechualdehyde spot and R f value is near 0.45, and the protocatechualdehyde spot separates well in the test sample chromatogram, and front and back are noiseless;
Method 3: developping agent is benzene-ethyl acetate-formic acid (18: 5: 0.8), and polarity is on the low side, and the protocatechualdehyde spot does not separate with the front and back spot in the test sample chromatogram.
Confirm that the optimum thin-layer developing agent of protocatechualdehyde is benzene-ethyl acetate-formic acid (8: 5: 0.8) in the separating traditional Chinese medicine composite preparation.
2.6 the selection of developer:
Draw each 5 μ l of need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, launch with following developping agent respectively, take out, dry, develop the color with following three kinds of developers.
Experimental technique and result are following:
Method 1: developer is that iodine vapor is smoked, and the colour developing that is badly specked of protocatechualdehyde spot and front and back should not be used for discriminating in the test sample chromatogram;
Method 2: developer is 1% liquor ferri trichloridi-1% potassium ferricyanide (1: 1), protocatechualdehyde clear spot in the test sample chromatogram;
Method 3: developer is the 5% vanillic aldehyde concentrated sulphuric acid, and 105 ℃ are heated to the spot colour developing.Though can be to aldehydes composition colour developing, owing to have transforming relationship between protocatechualdehyde and the protocatechuic acid, this developer after the colour developing, needs 105 ℃ of stability of protocatechualdehyde that added heat damage.Protocatechualdehyde spot and front and back spot are unintelligible in the test sample chromatogram.
Confirm that the optimum thin layer developer of protocatechualdehyde is 1% liquor ferri trichloridi-1% potassium ferricyanide in the separating traditional Chinese medicine composite preparation.
To sum up test is last confirms that the thin-layer chromatography discrimination condition of the red sage root in the Chinese medicinal composition preparation is:
Get present composition preparation 10g, porphyrize adds water 40ml and makes dissolving, and adding watery hydrochloric acid adjusting pH value is 3～4, extracts secondary with the ethyl acetate jolting; Each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methyl alcohol 1ml makes dissolving; As need testing solution, other gets the protocatechualdehyde reference substance, adds methyl alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution; According to the thin-layered chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be developping agent with benzene-ethyl acetate-formic acid (8: 5: 0.8); Launch, take out, dry, spray is with 1% liquor ferri trichloridi-1% potassium ferricyanide (1: 1).In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color.
Experimental example 6: earthworm thin layer discrimination test
Effective constituent in the present composition preparation in the contained earthworm medicinal material is the adenosine constituents.The present invention utilizes thin-layer chromatography to differentiate the earthworm medicinal material in the preparation.
1, test sample preparation method's selection
1.1 the selection of method for distilling
Experimental technique and result are following:
Get three parts of present composition preparation 10g, porphyrize adds chloroform 20ml respectively, close plug, and sonicated 20 minutes, reflow treatment 30 minutes, jolting were handled 60 minutes respectively, filtered, and filtrating is concentrated into 1ml, as need testing solution 1-3.
Get earthworm control medicinal material 1g, sonicated 20 minutes filters, and filtrating is concentrated into 1ml, processes control medicinal material solution.
Drawing each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, is developping agent with benzene-ethyl acetate (9: 1), launches, and takes out, and dries, and to ultraviolet lamp (365nm), inspects.
The result shows:
The jolting method can not be extracted fully, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, do not show the fluorescence spot of same color.Ultrasonic method and circumfluence method effect differ less, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, all show the fluorescence spot of same color.Because ultrasonic method is easier, select for use ultrasonic method to extract earthworm composition in the preparation.
2.2 the investigation of ultrasonic extraction
Adopt L
93
(4)The orthogonal experiment design is to three factors of influence extraction; Extraction solvent types, extraction solvent load, extraction time are investigated.See table 14.
Table 14
Through 9 parts of need testing solutions of above-mentioned experimental program preparation.
Get earthworm control medicinal material 1g, sonicated 20 minutes filters, and filtrating is concentrated into 1ml, processes control medicinal material solution.
Drawing each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, is developping agent with benzene-ethyl acetate (9: 1), launches, and takes out, and dries, and to ultraviolet lamp (365nm), inspects.
Experimental result is following:
When adopting sherwood oil to make extraction solvent, owing to contain the water of very small amount in the solvent, extraction efficiency reduces.In the test sample chromatogram with reference substance chromatogram relevant position on, do not have corresponding spot.
When adopting ethyl acetate to make extraction solvent, extracting power is strong excessively, and a lot of other compositions in the earthworm also are extracted out, in the test sample chromatogram, with reference substance chromatogram relevant position on the spot of apparent same color is arranged, but impurity is more, disturbs and differentiates.
When adopting chloroform to make solvent, extracting power is suitable, and relatively factors such as consumption, number of times and water consumption find to add chloroform 20ml (2 times of amounts), and sonicated was extracted fully in 20 minutes.Behind the need testing solution point sample of preparation, in the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color, spot is the most clear, noiseless.
Confirm that finally the preparation method who gets the earthworm need testing solution is: get present composition preparation 10g porphyrize, add chloroform 20ml, close plug, sonicated 20 minutes filters, and filtrating is concentrated into 1ml, as need testing solution.
3, the investigation of need testing solution point sample amount:
Draw need testing solution 2,5,8 μ l points respectively on same silica G plate, launch, examine and know.Experimental result is following:
When need testing solution point sample amount was 2 μ l, spot intensity was lower, should not observe; When need testing solution point sample amount was 5 μ l, clear spot on the thin layer was of moderate size, and did not have hangover, and front and back are noiseless; When need testing solution point sample amount was 8 μ l, spot was bigger on the need testing solution thin layer, linked to each other with front and back fluorescence spot.
The best point sample amount of confirming need testing solution is 5 μ l.
4, the selection of developping agent
According to the thin-layered chromatography test, draw need testing solution, each 5 μ l of control medicinal material solution, put respectively on same silica gel g thin-layer plate, with three kinds of methods expansion once, take out, dry, to ultraviolet lamp (365nm), inspect.
Experimental technique and result are following:
Method 1: developping agent is that benzene-ethyl acetate (2: 1) is a developping agent, and polarity is too small, and the spot Rf value is less in test sample and the control medicinal material chromatogram, does not separate with the front and back spot.
Method 2: developping agent is that benzene-ethyl acetate (9: 1) is a developping agent, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color, and spot is more suitable than transplanting.
Method 3: developping agent is for being that benzene-ethyl acetate (1: 1) is a developping agent, and polarity is excessive, and the spot Rf value is bigger in test sample and the control medicinal material chromatogram, does not separate with the front and back spot.
Confirm that the optimum thin-layer developing agent of earthworm composition is benzene-ethyl acetate (9: 1) in the separating traditional Chinese medicine composite preparation.
5, the selection of coloration method
Draw need testing solution, each 5 μ l of control medicinal material solution, put respectively on same silica gel g thin-layer plate, repeats three blocks of thin layer plates and launch respectively, taking-up is dried, below three kinds of modes develop the color.
Experimental technique and result are following:
Method 1: developer is that iodine vapor is smoked, is badly specked before and after the control medicinal material characteristic spot in the test sample chromatogram, should not be used for differentiating;
Method 2: inspect under colour developing mode to the ultraviolet lamp (365nm), in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color.
Method 3: developer is 10% sulfuric acid ethanol, and 105 ℃ are heated to the spot colour developing.Be badly specked before and after the control medicinal material characteristic spot in the test sample chromatogram, should not be used for differentiating;
Confirm that the optimum thin layer colour developing mode of earthworm composition is to inspect under the ultraviolet lamp (365nm) in the separating traditional Chinese medicine composite preparation.
To sum up test is last confirms that the thin-layer chromatography discrimination condition of earthworm in the Chinese medicinal composition preparation is:
Get present composition preparation 10g, porphyrize adds chloroform 20ml, close plug, ultrasonic place 20 minutes; Filter, filtrating is concentrated into 1ml, and as need testing solution, other gets earthworm control medicinal material 1g, shines medicinal material solution in pairs with legal system; According to the thin-layered chromatography test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be developping agent with benzene-ethyl acetate (9: 1); Launch, take out, dry, to ultraviolet lamp (365nm), inspect.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color.
Embodiment
Embodiment 1
Tangerine leaf 412.5g red sage root 412.5g spina gleditsiae 275g
Seed of cowherb 275g Fructus meliae toosendan 275g earthworm 275g
Above Six-element, except that earthworm, the seed of cowherb, four flavor boiling secondaries such as all the other tangerine leaves, each 1 hour, collecting decoction filtered, and it is 1.25～1.30 (85 ℃) that filtrating is concentrated into relative density, puts cold, subsequent use; Earthworm, the seed of cowherb 2 hours for the first time, 1 hour for the second time, filter with 70% alcohol reflux secondary; Merging filtrate adds in the above-mentioned concentrate, and the adjustment amount of alcohol reaches 70%, stirs; Leave standstill, reclaim ethanol and also be condensed into thick paste, an amount of with sucrose 500g and starch, dextrin, mixing; Process particle, drying is processed 1000g, promptly gets.
[discriminating]
(1) get these article 10g, porphyrize adds water 40ml and makes dissolving, and adding watery hydrochloric acid adjusting pH value is 3～4, extracts secondary with the ethyl acetate jolting; Each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methyl alcohol 1ml makes dissolving; As need testing solution, other gets the protocatechualdehyde reference substance, adds methyl alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution; According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be developping agent with benzene-ethyl acetate-formic acid (8: 5: 0.8); Launch, take out, dry, spray is with 1% liquor ferri trichloridi-1% potassium ferricyanide (1: 1).In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color.
(2) get these article 10g, porphyrize adds chloroform 20ml, close plug, sonicated 20 minutes; Filter, filtrating is concentrated into 1ml, and as need testing solution, other gets earthworm control medicinal material 1g, shines medicinal material solution in pairs with legal system; According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be developping agent with benzene-ethyl acetate (9: 1); Launch, take out, dry, to ultraviolet lamp (365nm), inspect.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color.
[assay]:
According to high performance liquid chromatography, chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filling agent; Methyl alcohol-0.025mol/L phosphoric acid solution (85% phosphoric acid solution 1.70ml is diluted with water to nearly 1000ml, and about 1.8ml regulates pH to 2.9～3.1 with triethylamine, adds water to 1000ml) (40: 60) is moving phase; The detection wavelength is 284nm.Number of theoretical plate calculates by the aurantiamarin peak should be not less than 5000.
The preparation of reference substance solution: it is an amount of that precision takes by weighing the aurantiamarin reference substance, adds methyl alcohol and process the solution that every 1ml contains 5 μ g, promptly gets.The preparation of need testing solution: get the content under these article content uniformity item, porphyrize is got 0.5g, and accurate the title decides, and puts in the conical flask; Add the about 40ml of methyl alcohol, sonicated (power 250W, frequency 33KHZ) 30 minutes is put coldly, filters; Filtrating is put in the 50ml measuring bottle, and filter residue and container be with methanol wash 2 times, 4ml at every turn, and washing lotion is filtered in the same measuring bottle, adds methyl alcohol to scale; Shake up, filter, get subsequent filtrate, promptly get.Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, and promptly get.These article contain the tangerine leaf with aurantiamarin (C for every bag
28H
34O
15) meter, must not be less than 3.0mg
[function with cure mainly] dispersing stagnated hepatoqi, promoting blood circulation and removing blood stasis, dissolving breast mass.Be used for stagnation of liver qi, qi depression to blood stasis, the proliferation of mammary gland, swollen breasts.
[usage and consumption] boiling water is taken after mixing it with water, one time 1 bag, 3 times on the one or follow the doctor's advice.
[specification] every packed 10g
Embodiment 2
Tangerine leaf 412.5g red sage root 412.5g spina gleditsiae 275g
Seed of cowherb 275g Fructus meliae toosendan 275g earthworm 275g
Above Six-element, except that earthworm, the seed of cowherb, four flavor boiling secondaries such as all the other tangerine leaves, each 1 hour, collecting decoction filtered, and it is 1.25～1.30 (85 ℃) that filtrating is concentrated into relative density, puts cold, subsequent use; Earthworm, the seed of cowherb 2 hours for the first time, 1 hour for the second time, filter with 70% alcohol reflux secondary; Merging filtrate adds in the above-mentioned concentrate, and the adjustment amount of alcohol reaches 70%, stirs; Leave standstill, reclaim ethanol and also be condensed into thick paste, an amount of with sucrose 500g and starch, dextrin, mixing; Process particle, drying is processed 1000g, promptly gets.
Assay:
According to high performance liquid chromatography, chromatographic condition and system suitability test use octadecylsilane chemically bonded silica to be filling agent; Methyl alcohol-0.025mol/L phosphoric acid solution (85% phosphoric acid solution 1.70ml is diluted with water to nearly 1000ml, and about 1.8ml regulates pH to 2.9～3.1 with triethylamine, adds water to 1000ml) (40: 60) is moving phase; The detection wavelength is 284nm.Number of theoretical plate calculates by the aurantiamarin peak should be not less than 5000.
The preparation of reference substance solution: it is an amount of that precision takes by weighing the aurantiamarin reference substance, adds methyl alcohol and process the solution that every 1ml contains 5 μ g, promptly gets.The preparation of need testing solution: get the content under these article content uniformity item, porphyrize is got 0.5g, and accurate the title decides, and puts in the conical flask; Add the about 40ml of methyl alcohol, sonicated (power 250W, frequency 33KHZ) 30 minutes is put coldly, filters; Filtrating is put in the 50ml measuring bottle, and filter residue and container be with methanol wash 2 times, 4ml at every turn, and washing lotion is filtered in the same measuring bottle, adds methyl alcohol to scale; Shake up, filter, get subsequent filtrate, promptly get.Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, and promptly get.These article contain the tangerine leaf with aurantiamarin (C for every bag
28H
34O
15) meter, must not be less than 3.0mg embodiment 3
Tangerine leaf 412.5g red sage root 412.5g spina gleditsiae 275g
Seed of cowherb 275g Fructus meliae toosendan 275g earthworm 275g
Above Six-element, except that earthworm, the seed of cowherb, four flavor boiling secondaries such as all the other tangerine leaves, each 1 hour, collecting decoction filtered, and it is 1.25～1.30 (85 ℃) that filtrating is concentrated into relative density, puts cold, subsequent use; Earthworm, the seed of cowherb 2 hours for the first time, 1 hour for the second time, filter with 70% alcohol reflux secondary; Merging filtrate adds in the above-mentioned concentrate, and the adjustment amount of alcohol reaches 70%, stirs; Leave standstill, reclaim ethanol and also be condensed into thick paste, an amount of with sucrose 500g and starch, dextrin, mixing; Process particle, drying is processed 1000g, promptly gets.
[discriminating]
(1) get these article 10g, porphyrize adds water 40ml and makes dissolving, and adding watery hydrochloric acid adjusting pH value is 3～4, extracts secondary with the ethyl acetate jolting; Each 30ml merges ethyl acetate liquid, and evaporate to dryness, residue add methyl alcohol 1ml makes dissolving; As need testing solution, other gets the protocatechualdehyde reference substance, adds methyl alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution; According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be developping agent with benzene-ethyl acetate-formic acid (8: 5: 0.8); Launch, take out, dry, spray is with 1% liquor ferri trichloridi-1% potassium ferricyanide (1: 1).In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color.
(2) get these article 10g, porphyrize adds chloroform 20ml, close plug, ultrasonic place 20 minutes; Filter, filtrating is concentrated into 1ml, and as need testing solution, other gets earthworm control medicinal material 1g, shines medicinal material solution in pairs with legal system; According to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B) test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, be developping agent with benzene-ethyl acetate (9: 1); Launch, take out, dry, to ultraviolet lamp (365nm), inspect.In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color.
Claims (4)
1. dispersing stagnated hepatoqi, the detection method of drug combination preparation promoting blood circulation and removing blood stasis is characterized in that this method comprises the steps:
A, assay: chromatographic condition and system suitability test, use octadecylsilane chemically bonded silica to be filling agent; 30-50: the methyl alcohol of 50-70 ratio-0.01～0.03mol/L phosphoric acid solution is a moving phase; The detection wavelength is 280～290nm; The preparation of reference substance solution: it is an amount of that precision takes by weighing the aurantiamarin reference substance, adds methyl alcohol and process the solution that every 1ml contains 2～10 μ g; The preparation of need testing solution: get said composition preparation porphyrize, get 0.5g, the accurate title, decide, and puts in the conical flask, adds the about 20～80ml of methyl alcohol; Sonicated 20～40 minutes is put coldly, filters, and filtrating is put in 25～100ml measuring bottle; Filter residue and container be with methanol wash 1～3 time, each 3～10ml, and washing lotion is filtered in the same measuring bottle, adds methyl alcohol to scale; Shake up, filter, get subsequent filtrate, promptly get; Determination method: accurate respectively reference substance solution and each 10～20 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly get, the composite preparation that is equivalent to crude drug 16-22g contains the tangerine leaf in aurantiamarin, must not be less than 3.0mg;
B, discriminating: get said composition preparation 10g, porphyrize adds water 30～50ml and makes dissolving, and adding watery hydrochloric acid adjusting pH value is 3～4; Extract 1～3 time with the ethyl acetate jolting, each 20～50ml merges ethyl acetate liquid; Evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the protocatechualdehyde reference substance, adds methyl alcohol and processes the solution that every 1ml contains 1～3mg, as reference substance solution; Draw each 2～10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with 4-12: 4-7: the benzene-ethyl acetate of 0.8 ratio-formic acid mixed solvent is a developping agent; Launch; Take out, dry, spray was with 1% liquor ferri trichloridi-1% potassium ferricyanide 1: 1; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;
C, discriminating: get said composition preparation 10g, porphyrize adds chloroform 10～30ml, close plug, and sonicated 15～30 minutes filters, and filtrating is concentrated into 1ml, as need testing solution; Other gets earthworm control medicinal material 1g, shines medicinal material solution in pairs with legal system, according to the thin-layered chromatography test; Draw each 2～10 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with 13-6: the benzene of 1 ratio-ethyl acetate mixed solvent is a developping agent; Launch; Take out, dry, to the 365nm ultraviolet lamp, inspect; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color;
Wherein, the bulk drug of said pharmaceutical composition consists of:
Tangerine leaf 275～550 weight portions, the red sage root 275～550 weight portions, spina gleditsiae 137.5～412.5 weight portions, the seed of cowherb 137.5～412.5 weight portions, Fructus meliae toosendan 137.5～412.5 weight portions, earthworm 137.5～412.5 weight portions.
2. the detection method of drug combination preparation as claimed in claim 1, this method comprises the steps:
Assay: chromatographic condition and system suitability test, use octadecylsilane chemically bonded silica to be filling agent; 40: 60 methyl alcohol-0.025mol/L phosphoric acid solution is a moving phase; The detection wavelength is 284nm; Number of theoretical plate calculates by the aurantiamarin peak should be not less than 5000; The preparation of reference substance solution: it is an amount of that precision takes by weighing the aurantiamarin reference substance, adds methyl alcohol and process the solution that every 1ml contains 5 μ g, promptly gets; The preparation of need testing solution: get the said composition preparation, porphyrize is got 0.5g, and accurate the title decides, and puts in the conical flask; Add the about 40ml of methyl alcohol, power 250W, frequency 33KHZ sonicated 30 minutes is put coldly, filters; Filtrating is put in the 50ml measuring bottle, and filter residue and container be with methanol wash 2 times, 4ml at every turn, and washing lotion is filtered in the same measuring bottle, adds methyl alcohol to scale; Shake up, filter, get subsequent filtrate, promptly get; Determination method: accurate respectively reference substance solution and each 10 μ l of need testing solution of drawing, inject liquid chromatograph, measure, promptly get;
Red sage root thin layer is differentiated: get said composition preparation 10g, porphyrize adds water 40ml and makes dissolving, and adding watery hydrochloric acid adjusting pH value is 3～4; Extract secondary with the ethyl acetate jolting, each 30ml merges ethyl acetate liquid; Evaporate to dryness, residue add methyl alcohol 1ml makes dissolving, as need testing solution; Other gets the protocatechualdehyde reference substance, adds methyl alcohol and processes the solution that every 1ml contains 1mg, as reference substance solution; Drawing each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is developping agent with benzene-ethyl acetates of 8: 5: 0.8-formic acid, launches, and takes out, and dries, and spray was with 1% liquor ferri trichloridi-1% potassium ferricyanide 1: 1; In the test sample chromatogram, with the corresponding position of reference substance chromatogram on show the spot of same color;
The earthworm thin layer is differentiated: get said composition preparation 10g, porphyrize adds chloroform 20ml, close plug, and sonicated 20 minutes filters, and filtrating is concentrated into 1ml, and as need testing solution, other gets earthworm control medicinal material 1g, shines medicinal material solution in pairs with legal system; Drawing each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is developping agent with 9: 1 benzene-ethyl acetates, launches, and takes out, and dries, and to the 365nm ultraviolet lamp, inspects; In the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the fluorescence spot of same color.
3. according to claim 1 or claim 2 the detection method of drug combination preparation is characterized in that the bulk drug of this pharmaceutical composition consists of:
Tangerine leaf 412.5 weight portions, the red sage root 412.5 weight portions, spina gleditsiae 275 weight portions, the seed of cowherb 275 weight portions, Fructus meliae toosendan 275 weight portions, earthworm 275 weight portions.
4. according to claim 1 or claim 2 the detection method of drug combination preparation is characterized in that the preparation method of said drug combination preparation is:
Except that earthworm, the seed of cowherb, four flavor boiling secondaries such as all the other tangerine leaves, each 1 hour, collecting decoction filtered, and it is 1.25～1.30 that filtrating is concentrated into 85 ℃ of survey relative densities, puts cold, subsequent use; Earthworm, the seed of cowherb 2 hours for the first time, 1 hour for the second time, filter with 70% alcohol reflux secondary; Merging filtrate adds in the above-mentioned concentrate, and the adjustment amount of alcohol reaches 70%, stirs; Leave standstill, reclaim ethanol and also be condensed into thick paste, an amount of with sucrose 500 weight portions and starch, dextrin, mixing; Process particle, drying is processed promptly and is got.
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| CN1698714A (en) * | 2005-05-10 | 2005-11-23 | 叶耀良 | 'Rukuaixiao' preparation for treating mammary disease and its preparation process |
| CN101279000A (en) * | 2007-04-03 | 2008-10-08 | 北京亚东生物制药有限公司 | Preparation of Chinese medicine soft capsules for curing mammary gland disease and products thereof |
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| CN1698714A (en) * | 2005-05-10 | 2005-11-23 | 叶耀良 | 'Rukuaixiao' preparation for treating mammary disease and its preparation process |
| CN101279000A (en) * | 2007-04-03 | 2008-10-08 | 北京亚东生物制药有限公司 | Preparation of Chinese medicine soft capsules for curing mammary gland disease and products thereof |
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| 展晓日,史新元,张培，等.乳块消片高效液相指纹图谱研究.《中国实验方剂学杂志》.2008,第14卷(第4期),1－3. * |
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Effective date of registration: 20160226 Address after: 102600, Daxing District Zhongguancun science and Technology Park, Daxing pharmaceutical industry base, Tianhe West Road, No. 19, Beijing Patentee after: Beijing rich church Pharmaceutical Technology Co., Ltd. Address before: 102200, 8, Zhenxing Road, Zhongguancun science and Technology Park, Beijing, Changping District Patentee before: Beijing Asia-East Bio-Pharmaceutical Co., Ltd. |