CN101288700B

CN101288700B - Chinese medicinal composition and preparation method and detection method thereof

Info

Publication number: CN101288700B
Application number: CN2007100655554A
Authority: CN
Inventors: 廖军鲜
Original assignee: BEIJING LUERCAO MEDICAL SCI-TECH Co Ltd
Current assignee: INNER MONGOLIA TIANQI ZHONGMENG PHARMACEUTICAL CO., LTD.
Priority date: 2007-04-16
Filing date: 2007-04-16
Publication date: 2011-05-18
Anticipated expiration: 2027-04-16
Also published as: CN101288700A

Abstract

The invention discloses a Chinese medicine composite for curing menopause syndrome, a preparation method and quality control method thereof. The medicine comprises the following raw materials: prepared fleeceflower root, epimedium, dodder, bush knotweed root, root of herbaceous peony and szechuan lovage rhizome, and the preparation method is as follows: the epimedium, the dodder and the root of herbaceous peony are boiled in water and ethanol is added for alcohol sedimentation; 50-70 percent of ethanol reflux is used for extracting the prepared fleeceflower root, the bush knotweed root and the szechuan lovage rhizome; alcohol extract is selected to combine with the alcohol sedimentation supernatant, pressure is reduced and the product is dried, and lactose, magnesium stearate, aerosil and aspartame are added for dry pressing and pelletization. The invention adopts high-performance liquid chromatography to test the content of 2, 3, 5, 4-tetrahydroxy-stilbene-2-O-beta-D-glucoside. The Chinese medicine composite has good efficacy of curing menopause syndrome.

Description

A kind of Chinese medicine composition and preparation method thereof and detection method

Technical field

The present invention relates to a kind of Chinese medicine composition and preparation method thereof and detection method, particularly relate to a kind of Chinese medicine composition for the treatment of climacteric syndrome and preparation method thereof and detection method.

Background technology

Climacteric syndrome is meant before and after the postmenopausal women, because the ovarian function decline, the low be directed at of estrogen level is based on the symptom of autonomic nervous system dysfunction.The traditional Chinese medical science thinks because asthenia of renal qi, due to the yin and yang imbalance.According to domestic and international interrelated data statistics, the women in 45-50 year 90% all has the different clinical manifestation of degree.Along with the arrival of aging society, the women's day of climacteric syndrome is cumulative many, according to statistics, China in 2000 more than 50 years old the women be about 1.2 hundred million, to reach approximately 2.8 hundred million to the year two thousand thirty,, become medical science problem and social problem that the whole world is paid close attention to therefore to the control of climacteric syndrome.

For treatment of climacteric syndrome doctor trained in Western medicine Hormone Replacement Therapy commonly used.(HormonReplaccment Therapy, HRT) HRT has clinical efficacy preferably, but untoward reaction is more, has increased the danger of breast carcinoma, carcinoma of endometrium, thrombotic disease etc., thereby has limited its use to a great extent.How the traditional Chinese medical science by the guidance of Chinese medical theory, according to patient's clinical manifestation, controlled with dialectical opinion, usually be main rule with the kidney invigorating and essence nourishing, make it reach kidney essense abundance, equilibrium between yin and yang, QI and blood is transferred and to be reached, then the purpose of its sick spontaneous recovery: and adverse reaction of tcm and side effect are little.Therefore, clinical practice is extensive day by day.The Chinese patent medicine of relevant treatment climacteric syndrome and relevant disease, not following tens kinds of relating in the document.The commodity of the one-tenth listing that has, as the GENGNIANAN coated tablet of Tianjin Le Rentang production, the climacteric health that the Guangzhou white clouds are produced by pharmaceutical factory, the ZHENHELING PIAN that Chinese medicine three factories in Shanghai City produce, the ground granule of Hunan Medical University, it is found pleasure in, joins stilbene Hydrargyri Oxydum Rubrum sheet, Herba Cistanches the kidney invigorating ball, pacifies a year climacteric sheet, nourishing blood to tranquillize the mind sheet, more U.S. Yiganning capsule etc. as woman's Yiganning capsule, climacteric Menstruation-Regulating Capsule sheet, climacteric.Make a general survey of raising variety, to have contained the kidney warming sun, nourishing kidney-yin, the kidney invigorating essence, replenishing QI and blood, removing summer-heat conscience burning hot though its function cures mainly, and adapts to insufficiency of kidney-YANG, deficiency of the kidney yin, hyperactivity of YANG due to deficiency of YIN, insufficiency of vital energy and blood or cards such as hepatic and renal YIN deficiency, hyperactivity of hesrt-fire and liver-fire, and many effects are single.Right Chinese medicine is thought, though climacteric syndrome is its main pathomechanism to suffer from a deficiency of the kidney, but because differences such as body constitution, also many clinically apoplexy due to deficiency are prominent real, usually involve internal organs such as conscience spleen in lysis, be difficult to take a part for the whole Yin Yang balancing when its key is the kidney invigorating and essence nourishing, QI and blood regulating with a kind of Therapeutic Principle.

Summary of the invention

The object of the invention is to provide a kind of Chinese medicine composition for the treatment of climacteric syndrome;

The object of the invention also is to provide a kind of Chinese medicine composition preparation method for the treatment of climacteric syndrome;

The object of the invention also is to provide a kind of detection method for the treatment of the Chinese medicine composition of climacteric syndrome.

The present invention seeks to be achieved through the following technical solutions:

The Chinese medicine composition of treatment climacteric syndrome of the present invention is to be made by the crude drug of following weight ratio:

Radix Polygoni Multiflori Preparata 200-800 weight portion Herba Epimedii 200-700 weight portion

Semen Cuscutae 300-800 weight portion Rhizoma Polygoni Cuspidati 200-700 weight portion

Radix Paeoniae Alba 200-700 weight portion Rhizoma Chuanxiong 200-700 weight portion.

The Chinese medicine composition of treatment climacteric syndrome of the present invention is preferably as follows that the crude drug of weight ratio makes:

Radix Polygoni Multiflori Preparata 400-700 weight portion Herba Epimedii 300-600 weight portion

Semen Cuscutae 500-700 weight portion Rhizoma Polygoni Cuspidati 300-500 weight portion

Radix Paeoniae Alba 300-600 weight portion Rhizoma Chuanxiong 300-600 weight portion.

The above-mentioned raw materials optimum ratio is:

Radix Polygoni Multiflori Preparata 600 weight portion Herba Epimedii 400 weight portions

Semen Cuscutae 600 weight portion Rhizoma Polygoni Cuspidati 400 weight portions

The Radix Paeoniae Alba 400 weight portion Rhizoma Chuanxiongs 400 weight portions

The above-mentioned raw materials optimum ratio is:

Radix Polygoni Multiflori Preparata 650 weight portion Herba Epimedii 350 weight portions

Semen Cuscutae 650 weight portion Rhizoma Polygoni Cuspidati 350 weight portions

The Radix Paeoniae Alba 550 weight portion Rhizoma Chuanxiongs 350 weight portions.

The above-mentioned raw materials optimum ratio is:

Radix Polygoni Multiflori Preparata 550 weight portion Herba Epimedii 580 weight portions

Semen Cuscutae 500 weight portion Rhizoma Polygoni Cuspidati 460 weight portions

The Radix Paeoniae Alba 360 weight portion Rhizoma Chuanxiongs 460 weight portions

Compositions of the present invention technology adding adjuvant is routinely made clinical acceptable forms such as tablet, capsule, oral liquid, drop pill, spray, granule; Described adjuvant comprises solvent, disintegrating agent, correctives, antiseptic, coloring agent, binding agent, lubricant, substrate etc.

Combination dosage form preferred particulates of the present invention agent, the preferred lactose of granule adjuvant, magnesium stearate, micropowder silica gel, aspartame add the adjuvant of following proportioning again in the above-mentioned raw materials medicine when making granule:

Lactose 400-700 weight portion magnesium stearate 3-10 weight portion micropowder silica gel 1-6 weight portion aspartame 3-15 weight portion; Ratio of adjuvant is preferably:

Lactose 560 weight portion magnesium stearate 5 weight portion micropowder silica gels 2 weight portion aspartames 9 weight portions; Lactose 450 weight portion magnesium stearate, 8 weight portion micropowder silica gel 2 weight portion aspartames, 12 weight portions or lactose 700 weight portion magnesium stearate 5 weight portion micropowder silica gels 5 weight portion aspartames 6 weight portions.

The preparation method of the Chinese medicine composition granular preparation that the present invention is above-mentioned is:

Choose crude drug and adjuvant: Herba Epimedii, Semen Cuscutae, the Radix Paeoniae Alba decoct with water 2-4 time, and each 1-2 hour, amount of water was 12-16 a times of medical material amount; Collecting decoction, being concentrated into 40-60 ℃, to survey relative density be 1.04-1.05, adds ethanol and make medicinal liquid contain the alcohol amount to reach 40-60%, and liquid is crossed in 3-10 ℃ of cold preservation, filters, and the precipitate with ethanol supernatant is standby; The alcohol reflux of Radix Polygoni Multiflori Preparata, Rhizoma Polygoni Cuspidati, Rhizoma Chuanxiong usefulness 50-70% 2-3 time adds ethanol 5-7 at every turn and doubly measures, and backflow 1-3 hour, merge alcohol extract, filter, promptly get alcohol extract; Get alcohol extract and above-mentioned precipitate with ethanol supernatant and merge, reclaim ethanol, be concentrated into relative density 1.20-1.40 to there not being the alcohol flavor, 80-50 ℃ of drying under reduced pressure, dry extract was pulverized 100 mesh sieves; Add lactose, magnesium stearate, micropowder silica gel, aspartame mixing, dry-pressing is granulated, promptly.

Drug regimen object detecting method of the present invention comprises one or more in following discrimination method and/or the assay:

Differentiate:

A. get the 1/10-2/5 unit formulation, add ethanol 20mL, supersound process 15-30 minute, filter, filtrate evaporate to dryness, residue add water 10mL dissolving, with ethyl acetate extraction 2-3 time, each 20mL, combined ethyl acetate liquid, evaporate to dryness, residue add water 10mL dissolving, cross polyamide column, the water 50mL of elder generation eluting discards water liquid, reuse 60-75% ethanol 50mL eluting, collect the eluent evaporate to dryness, residue adds methanol 2mL dissolving, as need testing solution; Other gets the icariin reference substance, adds methanol and makes the methanol solution that every 1mL contains 0.5mg, in contrast product solution; Test according to thin layer chromatography, draw each 4 μ L of need testing solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with 6-8: 1-3: lower floor's solution of 1-2 chloroform-methanol-water is developing solvent, saturated 10-20 minute, launch 9cm, take out, dry, spray is with 1% aluminum chloride alcoholic solution, place after 20-40 minute, put under the ultra-violet lamp and inspect; In the test sample chromatograph, with contrast chromatograph relevant position on, show the speckle of same color fluorescence;

B, get the 1/10-2/5 unit formulation, add chloroform 20mL, supersound process 5-15 minute, filter, filtrate evaporate to dryness, residue add methanol 1mL makes dissolving, as need testing solution; Other gets Rhizoma Polygoni Cuspidati control medicinal material 50-70 order 1g, adds chloroform 20mL, and supersound process 5-15 minute, filter, filtrate evaporate to dryness, residue add methanol 1mL makes dissolving, makes the 1mL methanol solution, in contrast product medical material solution; According to the thin layer chromatography test, draw need testing solution, each 4 μ L of control medicinal material solution, put respectively on same silica gel g thin-layer plate, with 8-11: 1-2: 0.1-0.3 toluene-ethyl acetate-formic acid is developing solvent, presaturation 10-20 minute, launches 9cm, take out, dry, under visible light, inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical yellow spotting;

C, get the 1/10-2/5 unit formulation, the 20mL that adds diethyl ether, supersound process 15-25 minute, filter, filtrate evaporate to dryness, residue add ethyl acetate 5mL makes dissolving, as need testing solution; Other gets Rhizoma Chuanxiong control medicinal material 50-70 order 0.5g, the 20mL that adds diethyl ether, and supersound process 15-25 minute, filter, filtrate evaporate to dryness, residue add ethyl acetate 5mL makes dissolving, makes the 5mL ethyl acetate solution, in contrast medical material solution; Test according to thin layer chromatography, draw each 8 μ L of need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, 60-90 ℃ of petroleum ether-ethyl acetate with 8-11: 0.4-0.7 is developing solvent, presaturation 10-20 minute, launch 9cm, take out, dry, put under the 365nm ultraviolet light and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the bright fluorescence speckle of same color;

D, get the 1/10-2/5 unit formulation, add methanol 20mL, supersound process 5-15 minute, filter, filtrate evaporate to dryness, residue add water 10mL makes dissolving, 3000 rev/mins of centrifugal 5-15 clocks are got supernatant and are crossed macroporous resin column, first water 50mL eluting, discard water liquid, reuse 25-35% ethanol 50mL eluting is collected eluent, evaporate to dryness, residue add water 10mL dissolving, use ether extraction 2-4 time, each 20mL, discard ether liquid, water liquid evaporate to dryness, residue adds the 5mL dissolve with methanol, cross the neutral alumina post, use the 20mL methanol-eluted fractions, collect eluent, evaporate to dryness, residue adds methanol 1mL makes dissolving, as need testing solution; Other gets the peoniflorin reference substance, adds ethanol and makes the solution that every 1mL contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw need testing solution 10 μ L, reference substance solution 4 μ L, put respectively on same silica gel g thin-layer plate, with 35-45: 4-7: 8-12: the solution of 0.1-0.8 chloroform-ethyl acetate-methanol-formic acid is developing solvent, presaturation 10-25 minute, launches 9cm, take out, dry, spray is with 5% vanillin sulfuric acid solution, and 100 ℃ are heated to the speckle colour developing; In the test sample chromatograph, with reference substance chromatograph relevant position on, show the speckle of same color;

Assay:

According to high performance liquid chromatography, chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler; 20-35: 65-80 methanol-0.2% phosphoric acid solution is a mobile phase; The detection wavelength is 320nm; Number of theoretical plate is by 2,3,5, and 4 '-tetrahydroxystilbene-2-O-β-D-glucoside peak calculates should be not less than 3000;

The preparation of reference substance solution: precision takes by weighing 2,3, and 5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside reference substance 5mg, put in the 50mL measuring bottle, add the 40-60% dissolve with methanol, and be diluted to scale, shake up, precision is measured 4mL, puts in the 25mL measuring bottle, adds 40-60% methanol and is diluted to scale, shake up, promptly get every 1mL and contain 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside 16 μ g;

The preparation of need testing solution: get drug combination preparation of the present invention, 70-90 order porphyrize is got the 1/30-1/10 unit formulation, and accurate the title decides, put in the tool plug conical flask, the accurate methanol 25mL that adds,, claim to decide weight, supersound process 15-30 minute, put coldly, claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, get subsequent filtrate 4mL, put in the 10mL measuring bottle, thin up is to scale, shake up, filter with 0.5 μ m microporous filter membrane, promptly; Algoscopy: respectively accurate each the 20 μ L of reference substance solution and need testing solution that draw, inject chromatograph of liquid, mensuration, that is, and contain Radix Polygoni Multiflori Preparata with 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside (C ₂₀H ₂₂O ₉) meter, must not be less than the 10-15mg/ unit formulation.

Drug regimen object detecting method of the present invention is preferably as follows one or more in discrimination method and/or the assay:

Differentiate:

A. get present composition granule, 60 order porphyrizes take by weighing 1/5 unit formulation, add ethanol 20mL, supersound process 20 minutes filters, and filtrate evaporate to dryness, residue add water 10mL dissolving, with ethyl acetate extraction 2 times, each 20mL, combined ethyl acetate liquid, evaporate to dryness, residue adds water 10mL dissolving, crosses 2g, 14-30 order, internal diameter 1.8cm polyamide column, the water 50mL of elder generation eluting discards water liquid, reuse 70% ethanol 50mL eluting, collect the eluent evaporate to dryness, residue adds methanol 2mL dissolving, as need testing solution; Other gets the icariin reference substance, adds methanol and makes the methanol solution that every 1mL contains 0.5mg, in contrast product solution; Test according to thin layer chromatography, draw each 4 μ L of need testing solution and reference substance solution, putting respectively on same silica gel g thin-layer plate, is developing solvent with lower floor's solution of 7: 2.5: 1 chloroform-methanol-water, saturated 15 minutes, launch 9cm, take out, dry, spray is with 1% aluminum chloride alcoholic solution, place after 30 minutes, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with contrast chromatograph relevant position on, show the speckle of same color fluorescence;

B, get present composition granule, 600 order porphyrizes take by weighing 1/5 unit formulation, add chloroform 20mL, and supersound process 10 minutes filters, and filtrate evaporate to dryness, residue add methanol 1mL makes dissolving, as need testing solution; Other gets Rhizoma Polygoni Cuspidati control medicinal material 60 order 1g, makes the 1mL methanol solution with method, in contrast product medical material solution; According to the thin layer chromatography test, draw need testing solution, each 4 μ L of control medicinal material solution, put respectively on same silica gel g thin-layer plate, with 9: 1: 0.2 toluene-ethyl acetate-formic acid was developing solvent, and presaturation 15 minutes launches 9cm, take out, dry, under visible light, inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical yellow spotting;

C, get present composition granule, 60 order porphyrizes take by weighing 1/5 unit formulation, the 20mL that adds diethyl ether, and supersound process 20 minutes filters, and filtrate evaporate to dryness, residue add ethyl acetate 5mL makes dissolving, as need testing solution; Other gets Rhizoma Chuanxiong control medicinal material 60 order 0.5g, makes the 5mL ethyl acetate solution with method, in contrast medical material solution; According to the thin layer chromatography test, draw each 8 μ L of need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with 9.5: 0.5 petroleum ether (60-90 ℃)-ethyl acetates was developing solvent, and presaturation 15 minutes launches 9cm, take out, dry, put under the 365nm ultraviolet light and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the bright fluorescence speckle of same color;

D; get present composition granule, 60 order porphyrizes take by weighing 1/5 unit formulation; add methanol 20mL, and supersound process 10 minutes filters; filtrate evaporate to dryness, residue add water 10mL makes dissolving, 3000 rev/mins of centrifugal 10 clocks; get supernatant and cross HPD100, high 8cm, internal diameter 1cm macroporous resin column; first water 50mL eluting, discards water liquid, reuse 30% ethanol 50mL eluting; collect eluent, and evaporate to dryness, residue add water 10mL dissolving; use ether extraction 3 times, and each 20mL discards ether liquid; water liquid evaporate to dryness, and residue adds the 5mL dissolve with methanol, crosses 2g; internal diameter 1cm neutral alumina post, uses the 20mL methanol-eluted fractions, collects eluent; evaporate to dryness, residue add methanol 1mL makes dissolving, as need testing solution; Other gets the peoniflorin reference substance, adds ethanol and makes the solution that every 1mL contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw need testing solution 10 μ L, reference substance solution 4 μ L, put respectively on same silica gel g thin-layer plate, with 40: 5: 10: the solution of 0.2 chloroform-ethyl acetate-methanol-formic acid was developing solvent, and presaturation 20 minutes launches 9cm, take out, dry, spray is with 5% vanillin sulfuric acid solution, and 100 ℃ (or hair dryer) is heated to the speckle colour developing; In the test sample chromatograph, with reference substance chromatograph relevant position on, show the speckle of same color;

Assay:

According to high performance liquid chromatography, chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler; Methanol-0.2% phosphoric acid solution was a mobile phase in 29: 71; The detection wavelength is 320nm; Number of theoretical plate is by 2,3,5, and 4 '-tetrahydroxystilbene-2-O-β-D-glucoside peak calculates should be not less than 3000; The preparation of reference substance solution: precision takes by weighing 2,3, and 5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside reference substance 5mg, put in the 50mL measuring bottle, add 50% dissolve with methanol, and be diluted to scale, shake up, precision is measured 4mL, puts in the 25mL measuring bottle, adds 50% methanol and is diluted to scale, shake up, promptly get every 1mL and contain 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside 16 μ g;

Present composition granule is got in the preparation of need testing solution, and 80 order porphyrizes are got 1/20 unit formulation, the accurate title, decide, and puts in the tool plug conical flask, and the accurate methanol 25mL that adds claims to decide weight, power 100W, frequency 40KHz supersound process 20 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, get subsequent filtrate 4mL, put in the 10mL measuring bottle, thin up is to scale, shake up, filter with 0.5 μ m microporous filter membrane, promptly; Algoscopy: respectively accurate each the 20 μ L of reference substance solution and need testing solution that draw, inject chromatograph of liquid, mensuration, that is, and contain Radix Polygoni Multiflori Preparata with 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside (C ₂₀H ₂₂O ₉) meter, must not be less than the 12.5mg/ unit formulation.

Described compositions detection method can be applied to the various dosage forms of compositions, as clinical acceptable forms such as tablet, capsule, oral liquid, drop pill, spray, granules, because the wherein contained suitable crude drug amount of preparation of different dosage form is identical, therefore each dosage form is when detecting, selected sample size can be unified conversion and be suitable crude drug amount, this detection method is the per unit preparation with suitable crude drug amount 15-20g, and the per unit preparation can be every, every, every or every ball etc.

The present composition is under instruction of Chinese Medicine theory, according to involutional pathogenesis and symptom feature, controls by dialectical opinion, develops on the basis in conjunction with clinical practice for many years.Its prescription is made up of medicines such as Radix Polygoni Multiflori Preparata, Herba Epimedii, Semen Cuscutae, Rhizoma Polygoni Cuspidati, the Radix Paeoniae Albas, has the kidney invigorating and essence nourishing, YIN and YANG balance regulating, and the effect that relieving restlessness is calmed the nerves gets final product kidney tonifying to effect a permanent cure, and relieving restlessness is calmed the nerves to take stopgap measures again.Through clinical verification for many years, prove this side's determined curative effect, and in the drug effect toxicologic study, be able to further proof.The present composition is safe in utilization, determined curative effect, and preparation is appropriate, and quality controllable, stable, curative effect is better compared with similar products, has excellent development potentiality and market prospect.The extraction of present composition preparation, separating technology condition, preparation and dosage form are reasonable, have set up pointed, handling stronger detection method and standard, and the quality of the pharmaceutical preparations is stable.

The present invention is directed to special physiological disease characteristics of climacteric, with Zhi Qiben, the relieving restlessness of calming the nerves is with Gu Qibiao with benefiting kidney-essence, YIN and YANG balance regulating, remove to reach this solid mark, negative and positive close and, its card is from the purpose of removing.Not only effective to involutional women, diseases such as vexed, insomnia, soreness of waist and knee joint are appearred in involutional male also extraordinary curative effect.Be used for before and after climacteric that deficiency of kidney-essence, disarmony between the heart and kidney cause syndrome and male for treatment and suffer from similar symptom significant curative effect is arranged, and safe and effective by drug effect, toxicity and clinical research proof we.Pharmacodynamic test of active extract shows, the granule that the present composition is made can improve the serum E2 level of female removal ovary rat climacteric model, reduce FSH and LH level, show that it has the improvement effect to sexual hormone disturbance, inductive sleep has certain synergism to pentobarbital sodium to make this animal pattern simultaneously, show as sleeping the quickening, dropping asleep latency shortens, and obviously prolong the length of one's sleep.The osteoporosis rat model that dexamethasone is caused, present composition granule can obviously increase the heavy and proportion of femur ash, obviously improve multinomial bone meterological index, use calcium level to recover normal simultaneously, similar to the effect of female rats and male rat.Bringing out the mice sweat secretion by pilocarpine increases, and the present composition has remarkable anti-hidropoiesis, and the sweating of normal mouse is not had obvious influence, can obviously prolong the normal mouse swimming with a load attached to the body time simultaneously.

Following experimental example and embodiment are used to further specify but are not limited to the present invention.

The influence of the osteoporosis rat that 1 pair of dexamethasone of experimental example causes

1, test objective: the osteoporosis rat model that adopts dexamethasone to cause, observe of the influence of present composition granule, whether osteoporosis is had the improvement effect to understand peaceful granule to rat bone proportion, density, bone ash weight and bone meterological index.

2, test material

2.1 test sample

2.1.1 be subjected to reagent: present composition granule, lot number: 20041206

2.1.2 source: dosage form chamber, institute of Chinese materia medica

2.1.3 content: 7.1g crude drug/g medicated powder

2.1.6 solvent: distilled water

2.1.7 compound method: with the peaceful granule medicated powder of the accurate weighing of electronic balance, adding distil water grind be mixed with concentration be 0.035,0.07 and 0.14g medicated powder/ml (be equivalent to respectively: 0.25,0.5, the 1.0g crude drug/ml) three kinds are tried concentration

2.2 positive drug:

2.2.1 title: XIANLING GUBAO JIAONANG

2.2.1 source: Tongjitang Pharmacy Co., Ltd., Guizhou

2.2.2 lot number: 041012

2.2.3 solvent: distilled water

2.2.4 specification: 0.5g/ grain

2.2.5 compound method: XIANLING GUBAO JIAONANG is made into 0.07g medicated powder/ml with distilled water

2.2.6 negative control product: distilled water

2.3 modeling medicine

2.3.1 title: dexamethasone injection

2.3.2 source: Shanghai the 9th pharmaceutical factory

2.3.3 lot number: 06251

2.3.4 solvent: saline

2.3.5 specification: 25mg/ml

236 compound methods: be diluted to 2.5mg/ml with normal saline

2.4 laboratory animal and raising

2.4.1 kind: rat

2.4.2 strain: SD

2.4.3 sex and quantity: totally 72, female, hero half and half

2.4.4 the weight of animals: 240-260g

2.4.5 animal origin: meeting sharp magnificent laboratory animal company limited by Beijing dimension provides, the quality certification number: SCXK capital 2002------2006

2.4.6 feedstuff: Chinese Military Medical Science Institute provides

2.4.7 raise: animal feeding in the SPV of China Academy of TCM level Animal House, 19-22 ℃ of temperature, humidity 40%-70%, artificial lighting, 12 hours light and shade cycles

2.4.8 every treated animal number: 12, female, male each

3. experimental technique

After rat arrived, adaptability was raised 3 days.Then rat is divided into 6 groups at random, 12 every group, female, male each 6.Be grouped into: (1) normal control group, (2) model group, (3) positive drug XIANLING GUBAO group 0.7g/kg, the high, medium and low dosage group of (4)～(6) present composition granule (10,5,2.5g crude drug/kg).Be subjected to the route of administration of reagent present composition granule and positive drug XIANLING GUBAO to be gastric infusion, consistent with approach with clinical plan, every day, gastric infusion was 1 time, and successive administration 50 days, administration volume are the 1ml/100g body weight.Matched group is irritated the isopyknic distilled water of stomach.From on-test, except matched group, all the other each group all gives dexamethasone 2 times in intramuscular injection weekly, and dosage is 2.5mg/kg, 2 dosing intervals 3 days, and one until off-test.In the 14th day of off-test with finish preceding the 3rd day, the equal lumbar injection of all test group gives quadracycline 30mg/kg (Sigma import packing).

All test group are fasting 12h after the last administration.Weigh, get blood, centrifugal, separation of serum is measured calcium, phosphorus and is contained, and with sacrifice of animal.

Get the bilateral femur,, pick clean soft tissue and muscle, with electronic balance weighing femur weight in wet base, measure the femur volume with the volume determination instrument.With baking box the bilateral femur is toasted 48h under 100 ℃ of temperature then, the dry femur weight of weighing.And then femur inserted in the muffle furnace, 500 ℃ of roasting 9h make it ashing.Weighing bone ash weight.

Get rats with left proximal tibia 1/3, remove soft tissue.Dewater step by step with ethanol, dimethylbenzene is transparent, every grade each twice, each 24h.Soak into the preparation of liquid: I liquid: methyl methacrylate (Beijing Yili Fine Chemicals Co., Ltd., lot number: 20050317) 75ml, dibutyl phthalate (Beijing chemical reagents corporation, lot number: 040401) 25ml; II liquid: on the basis of I liquid, add benzoyl peroxide (the imperial chemical reagent company limited of Beijing gold, lot number: 20000420) 1g; III liquid: on the basis of I liquid, add benzoyl peroxide 2.5g.More than three liquid, fully stir evenly with magnetic stirring apparatus.Specimen is respectively soaked into 36h at I, II, III liquid.In the penicillin bottle, inject the III liquid of about 5ml, specimen is put into bottle by same direction, place 40 ℃ of baking oven polyase 13～4 day then, wait to become colorless behind the transparent hard embedded block, smash bottle, the taking-up embedded block.After repairing piece, on Reicheit-Jung2040 microtome (Germany), cut out vertical undecalcified bone slice of 5 μ m and 10 μ m respectively with the wolfram steel cutter.After 5 μ m section dissolved away resin with dimethylbenzene, gradient ethanol carried out Toluidine blue staining to water, observed the bone trabecula form and carried out the observation of osseous tissue morphometry; Every tibia refabrication 10 μ m simultaneously directly give for 2 times behind the tetracycline at the sedimentary tetracycline fluorescence of sclerotin with fluorescence microscope, with the spacing between 2 sedimentary fluorescent belts as the cortical bone mineralization rate.

The mensuration of osseous tissue morphometry index, the method according to Zhang Ming such as puts at the people, adopt the LeicaQwin image analysis system to carry out following osseous tissue morphometry and measure:

The metering of bone trabecula tissue morphology:

Trabecula Bone Volume percentage ratio (TBV%): Trabecula Bone Volume accounts for the percentage ratio of tested medullary cavity cumulative volume, is the flat outstanding feature of bone water gaging:

Bone trabecula sorbent surface percentage ratio (TRS%): the percentage ratio on irregular, rough bone trabecula surface;

Bone trabecula forms surface percentage (TFS%): the OS that has osteoblast to be covered accounts for the percentage ratio on bone trabecula surface;

The active surface percentage (AVS%) that generates: have the bone trabecula surface of fluorescent labeling band to account for the percentage ratio on bone trabecula surface:

Bone trabecula mineralization rate (MAR): the natural law that the bone trabecula surface is separated by divided by twice labelling by the average distance of tetracycline fluorescence double labelling band.

Norphometry in the cortex:

Osteoid mean breadth (OSW): the mean breadth of cortex inner surface osteoid;

Cortical bone mineralization rate (mAR): the natural law that the average distance of 2 tetracycline fluorescence indicia band is separated by divided by twice labelling in the cortex.

4. statistical analysis

T check carrying out statistical analysis between the employing group, relatively the difference of each administration group and model group thinks that with P＜0.05 significant difference is arranged.

Femur proportion=femur dry weight/flesh bone volume

Femur ash proportion=femur ash weight/femur volume

5. result

5.1 influence to femur dry weight, ash weight and proportion thereof

Femur dry weight, ash weight and proportion thereof can reflect the density of femur.The result shows, after giving people Mus long term injections dexamethasone, rat femur dry weight and proportion thereof, the femur ash is heavy and the aggregatecement ratio weight average obviously reduces (with matched group P＜0.05 relatively, or P＜0.01), the bone density that reflects model group reduces, and illustrates to have tangible osteoporotic conditions.Irritate stomach and give the present composition granule 10g crude drug/kg, femur dry weight and bone proportion female, tom there is rising trend, can obviously increase the heavy and bone ash proportion (comparing p＜0.05) of femur ash, illustrate that present composition granule can obviously suppress the osteoporosis that dexamethasone causes with model group.The results are shown in Table 1-4

Table 1 present composition granule is to the influence (g) of the inductive osteoporosis rat femur of dexamethasone dry weight (X ± SD)

Annotate: compare * p＜0.05, * * p＜0.01 with model group

The influence (carried interest bone weight g/ femur volume ml) that table 2 present composition granule is heavy to the inductive osteoporosis rat thigh of dexamethasone anharmonic ratio (X ± SD)

Annotate: compare * p＜0.05, * * p＜0.01 with model group

The influence (g) that table 3 present composition granule is heavy to the inductive osteoporosis rat femur ash of dexamethasone (X ± SD)

Annotate: compare * p＜0.05, * * p＜0.01 with model group

Table 4 present composition granule is to the influence of the inductive osteoporosis rat femur of dexamethasone ash proportion (femur ash weight g/ femur volume ml, X ± SD)

Annotate: compare * p＜0.05, * * p＜0.01 with model group

5.2 separately or during joint account, the model group blood calcium all has reduction trend than normal group, but does not see statistical significance with the blood calcium of female, tom, phosphorus content.Show that dexamethasone has certain influence to calcium metabolism in the rat body, but because the animal compensatory capacity is stronger, its influence degree does not reach the significance,statistical meaning as yet.The blood calcium of present composition granule 2.5g, 5.0, female, the male calculating separately of 10.0g crude drug/kg dosage group or joint account all has the trend of increasing than model group, 2.5g crude drug/kg group has the significance,statistical meaning, the blood calcium value of other groups is all near the normal control group.The result shows that present composition granule can improve the calcium level that dexamethasone causes osteoporosis rat, makes it to recover normal.The results are shown in Table 5.

6. conclusion

Present composition granule 5～10g crude drug/kg, on the female and male rat osteoporosis model that dexamethasone causes, can obviously increase femur ash weight and proportion thereof and (compare P＜0.05=with model group, obviously improve multinomial bone meterological index, can also improve the calcium level that dexamethasone causes osteoporosis rat, make it to recover normal.Therefore, present composition granule improves significantly to the inductive female and equal tool of male rat osteoporosis of dexamethasone.Present composition granule is similar to the osteoporosis improvement effect of female rats and male rat.

Blood plasma sex gland hormones, calcitonin, Bone Gla protein and the gonad organ weight's of 2 pairs of female rats of experimental example climacteric model influence

Adopt removal ovary rat climacteric model, irritate stomach and give present composition granule 2.5,5,10g crude drug/kg, continuous 1 month, observe of the influence of present composition granule sex gland hormones, calcitonin, Bone Gla protein and gonad organ weight.The result shows that each dosage group of present composition granule does not have obvious influence to the gonad organ weight of removal ovary rat; 10g crude drug/kg has tangible castering action (comparing P＜0.05 with model group) to serum estradiol (E2) level of removal ovary rat, and rate of rise is that 32.17%:5g crude drug/kg has the trend of increasing to the E2 level, and the rate of increasing is 25.5%.The middle and high dosage group of present composition granule (5,10g crude drug/calcitonin kg), Bone Gla protein have rising trend than model group, the rate of rise of middle and high dosage group calcitonin is respectively 15.5% and 21.33%, and the rate of rise of Bone Gla protein is respectively 32.3% and 32.8%.The result shows that present composition granule can obviously improve body inner estrogen level, regulates calcitonin and Bone Gla protein level, suppresses bone resorption, improves the osteoblast activity, and osteoporosis is played certain improvement effect.

The influence of experimental example 3 pairs of male castrated rats plasma testosterones, Bone Gla protein, calcitonin and sexual organ's weight

Adopt male castrated rats, irritate stomach and give present composition granule 2.5,5,10g crude drug/kg, continuous 1 month, observe of the influence of present composition granule, androgen is reduced relevant pathophysiological change improvement effect whether to understand present composition granule to plasma testosterone, Bone Gla protein, calcitonin and gonad organ weight.The result shows that present composition granule does not have obvious influence to the testosterone level and the male organs weight of castrated rats, and its no androgen-like action is described.Present composition granule high dose group (10g crude drug/kg) calcitonin is had tangible rising effect (comparing P＜0.05 with model group), the rate of increasing is 55.2%; The blood Bone Gla protein level of 3 dosage groups all has certain rising trend, shows that present composition granule can suppress the osteoclast activity by the adjusting to calcitonin and Bone Gla protein, suppresses bone absorption, thereby osteoporosis is had the improvement effect.

The influence of 4 pairs of removal ovary rats of experimental example lengths of one's sleep

Adopt the rat of extracing ovary as the climacteric model, whether observation present composition granule has improvement effect to understand it to involutional insomnia to the influence of the length of one's sleep.The result shows that present composition granule 5,10g crude drug/kg can make the sleeping quickening of animal, and dropping asleep latency obviously shortens (comparing P＜0.05 with model group); Obviously prolong the length of one's sleep.The result shows that present composition granule has certain mitigation to involutional sleep disorder.

The influence of 5 pairs of normal spontaneous activity in mice of experimental example

Adopt normal mouse, irritate stomach and give present composition granule 3,6,12g/kg, every day 1 time, successive administration 7 days is observed the influence of present composition granule to spontaneous activity.The result shows that the spontaneous activity of each dosage group of present composition granule time number average is close with matched group, shows that present composition granule does not have obvious influence to normal spontaneous activity in mice.

6 pairs of mices of experimental example normally secrete antiperspirant and the ergogenic effect of the inductive sweat gland secretion of pilocarpine

Adopt the hyperfunction mouse model of the inductive juice glandular secretion of normal mouse and pilocarpine respectively, observe the anti-hidropoiesis of present composition granule.The result shows that on the hyperfunction model of the sweat secretion that pilocarpine causes, present composition granule 6,12g crude drug/kg show significant anti-hidropoiesis, but its sweat gland secretion to normal mouse does not have obvious influence.The result shows, present composition granule increases abnormal sweating and has anti-hidropoiesis.

The influence of 7 pairs of normal mouse swimming endurances of experimental example

Observed of the influence of present composition granule to normal mouse swimming with a load attached to the body endurance.The result shows, present composition granule 3,6g crude drug/kg dosage group gastric infusion continuous 7 are big, can obviously prolong the swimming with a load attached to the body time (with matched group P＜0.05 relatively) of mice, and tool prolongs percentage rate and is: 35.1-73.8%.The result shows that present composition granule has certain antifatigue effect.

Experimental example 8 extraction process Study on Conditions

1. Herba Epimedii, Semen Cuscutae, Radix Paeoniae Alba extraction process by water condition is preferred

(1) orthogonal test of Herba Epimedii, Semen Cuscutae, Radix Paeoniae Alba extraction process by water condition

Method: taken by weighing Herba Epimedii 16g, Herba cuscutae respectively 24g, Radix Paeoniae Alba 16g, totally 9 parts, press the experimental condition arrangement test of the factor level and the table 7 of table 6, add the water reflux, extract,, aqueous extract cold preservation is spent the night, and gets supernatant respectively after centrifugal, measures content Determination of Icariin with HPLC method (by the pharmacopeia method), and it is carried out variance analysis, the results are shown in Table 8.

Table 6 factor level table

Table 7 orthogonal test table

Table 8 analysis of variance table

*：F _1-0.05(2，2)＝19。0**：F _1-0.01(2，2)＝99.0

As shown in Table 8, A factor (amount of water) does not have remarkable influence to the result, A ₃Be optimum condition; B factor (extraction time) does not have the significance influence to the result, with B ₃Be the best; C factor (extraction time) does not have influence to the result, determines C ₃Be the best.So it is A that liquid medicine such as Herba Epimedii are carried optimum condition ₃B ₃C ₃, also promptly: decoction pieces adds 14 times in water, heating extraction 3 times, each 1.5 hours.

(2) water is carried part optimum extraction process replica test

Take by weighing Herba Epimedii 60g, Semen Cuscutae 90g Radix Paeoniae Alba 60g in the prescription ratio, amount to 210g, press three parts of optimum condition parallel extraction, measure icariin content, the results are shown in Table 9.

Table 9 water is put forward optimised process replica test result

By table 9 result as can be known, liquid medicine such as the Herba Epimedii that filters out are carried process stabilizing, and icariin content is higher than Orthogonal experiment results, illustrate that the extraction conditions that optimizes is suitable.

2. Radix Polygoni Multiflori Preparata, Rhizoma Polygoni Cuspidati, Rhizoma Chuanxiong ethanol extraction process conditions is preferred

(1) orthogonal test of Radix Polygoni Multiflori Preparata, Rhizoma Polygoni Cuspidati, Rhizoma Chuanxiong ethanol extraction

Method: take by weighing totally 9 parts of Radix Polygoni Multiflori Preparata 12g, Rhizoma Polygoni Cuspidati 8g, Rhizoma Chuanxiong 8g respectively, press the experimental condition arrangement test of the factor level and the table 11 of table 10, add different concentration ethanol and extract 2 times, liquid is crossed in extracting solution cold preservation, gets supernatant after centrifugal, concentrate drying, weigh, calculate paste-forming rate, and measure the content of stilbene glucoside with HPLC method (by the pharmacopeia method), carry out variance analysis, the results are shown in Table 12.

Table 10 factor level table

Table 11 orthogonal test table

Table 12 analysis of variance table

*：F _1-0.05(2，4)＝6.94?F _1-0.01(2，4)＝18.00

As shown in Table 12, A factor (concentration of alcohol) does not have remarkable influence to the result, and A2 is an optimum condition; C factor (extraction time) has a significant effect to the result, and C3 is best.Taking all factors into consideration the alcohol extraction optimum condition is A ₂B ₃C ₃, also promptly: decoction pieces adds 6 times of 60% ethanol, heating extraction 2 times, each 2 hours.

(2) comparison of Radix Polygoni Multiflori Preparata, Rhizoma Polygoni Cuspidati, Rhizoma Chuanxiong ethanol extraction number of times

Method: take by weighing Radix Polygoni Multiflori Preparata 60g, Rhizoma Polygoni Cuspidati 40g, Rhizoma Chuanxiong 40g respectively, add 6 times of extractions of 60% ethanol, each 2h, extraction time is pressed table 13 and is arranged test, and each time extracting solution cold preservation respectively spends the night, and gets supernatant after centrifugal, concentrate drying, weigh, measure the content of stilbene glucoside, and calculate the rate of transform of paste-forming rate and stilbene glucoside by the pharmacopeia method.The results are shown in Table 13.

The comparative result of table 13 Radix Polygoni Multiflori Preparata, Rhizoma Polygoni Cuspidati, Rhizoma Chuanxiong ethanol extraction number of times

By table 13 as seen, account for 83.93% of medical material content with total rate of transform of extracting for the second time stilbene glucoside for the first time.Consider that cost and environmental protection factor selective extraction number of times are 2 times.

(3) best alcohol extraction process replica test

Get Radix Polygoni Multiflori Preparata 45g, Rhizoma Polygoni Cuspidati 30g, Rhizoma Chuanxiong 30g, three parts, add 6 times of 60% ethanol and extract 2 times, to extract 2 hours at every turn, extracting solution cold preservation is spent the night, and gets supernatant after centrifugal, and concentrate drying is weighed, and measures the content of stilbene glucoside by the pharmacopeia method, and calculates paste-forming rate.The results are shown in Table 14.

Table 14 Radix Polygoni Multiflori Preparata, Rhizoma Polygoni Cuspidati, Rhizoma Chuanxiong alcohol extraction optimised process repeated trials result

By table 14 result as can be known, Radix Polygoni Multiflori Preparata, Rhizoma Polygoni Cuspidati, Rhizoma Chuanxiong alcohol extraction process are stablized, and stilbene glucoside content is higher than the orthogonal test best values.The alcohol extraction process of determining Rhizoma Polygoni Cuspidati, Rhizoma Chuanxiong thus is: 60% ethanol extracts 2 times for 6 times, extracts 2 hours at every turn.

Experimental example 9 separation, purification, concentrate and the research of drying process

The research of (one), separation, purifying process

1. Herba Epimedii Semen Cuscutae, Radix Paeoniae Alba water extract is refining

(1) comparative test of water extract alcohol precipitation concentration

Get Herba Epimedii, Semen Cuscutae, closing of the Radix Paeoniae Alba and fry in shallow oil concentrated medicament (the suitable 1g medical material of every ml) 50ml, three parts, add ethanol respectively and reach 50%, 60% and 70% respectively to containing the alcohol amount, cold preservation is spent the night, and gets supernatant and measures content Determination of Icariin.The results are shown in Table 15.

The comparative test result of table 15 alcohol precipitation concentration

By table 15 result as seen, influence is little to the precipitate with ethanol effect for concentration of alcohol, and icariin content is similar, considers production cost, so adopt 50% ethanol heavy.

The comparison of medicinal liquid concentrating degree during (2) 50% precipitate with ethanol

Get boil medicine three parts of liquid of closing of Herba Epimedii, Semen Cuscutae, the Radix Paeoniae Alba, concentration concentrates in the according to the form below respectively, measures relative density, adds ethanol and reaches 50% to containing the alcohol amount, and cold preservation is spent the night, and gets supernatant and measures content Determination of Icariin.The results are shown in Table 16.

The comparison of medicinal liquid concentrating degree during table 16 50% precipitate with ethanol

By table 16 result as seen, the concentrating degree of medicinal liquid has considerable influence to icariin content, so when selecting medicinal liquid to be concentrated into relative density 1.05 (suitable 0.5 medical material of every ml), add ethanol and make and contain the alcohol amount and reach 50% method and remove impurity.

(2) concentrate and drying process research

The extracting solution of getting the 600g medical material reclaims ethanol to there not being the alcohol flavor, is divided into 3 parts, and portion is concentrated into relative density 1.10, for reserve liquid 1..In addition 2 parts are concentrated into relative density 1.30, for reserve liquid 2. and 3..Get reserve liquid and 1. carry out spray drying (170 ℃ ± 5 ℃ of inlet temperatures, 75 ℃ ± 5 ℃ of outlet temperatures, pressure 1kg/cm ²) test, the difficulty of spray drying powder delivery as a result, glutinous wall phenomenon is serious.

Other gets 2. and 3. drying under reduced pressure under 50 ℃ and 70 ℃ of conditions respectively of reserve liquid, and dry thing is carried out the assay of stilbene glucoside.The results are shown in Table 17.

The different baking temperatures of table 17 are to the influence of stilbene glucoside content in the extract

The result shows that extractum easily swells during drying under reduced pressure, and drying effect is better, and dry extract is loose, helps operations such as next step pulverizing, granulation.Baking temperature is very little to the stilbene glucoside content influence, is concentrated into relative density 1.30 so adopt, and controls 70 ℃ of baking temperatures, below carries out the drying process of drying under reduced pressure.

Experimental example 10 preparations shaping technical studies

1, the selection of method of granulating

Once tested this product and use the spray drying one-step palletizing, because the adjuvant amount is more less than just, wall sticking phenomenon is serious.Adopt dry granulation then can granulate smoothly, can also save the energy simultaneously, reduce cost, so the method for granulating of this product has been selected dry granulation.Result of the test proves that the dry granulation effect is better, has reduced supplementary product consumption, and grain graininess is even, and is aesthetic in appearance.

2, granulate with the selection of adjuvant kind and consumption

(1) filler breed selection

Method: it is an amount of to get extract, respectively adjuvants such as pregelatinized Starch, lactose, dextrin carried out preferably, and serve as to investigate index with grain forming rate and moisture absorption percentage rate, supplementary product kind, consumption and granulation situation see Table 18.

Table 18 filler breed selection

Annotate: "-" be not for surveying.

Mouldability is bad when using pregelatinized Starch (No. 2), dextrin (No. 3) and mixed accessories (No. 4) to do the filler granulation as known from Table 18, so do not adopt; And it is more suitable to make filler with lactose (No. 1).

(2) selection of fluidizer, lubricant kind and consumption

In order to improve the further flowability of improving molding particles situation and drug powder, again fluidizer and lubricant kind and consumption are selected.Method: it is an amount of to get extract, adds the lactose mixing, adds different fluidizer and lubricant respectively, and mixed pelletization the results are shown in Table 19.

The selection of table 19 fluidizer and lubricant kind and consumption

By table 19 as seen, the grain forming rate and the granulation situation of 3 kinds of prescriptions are all relatively good, select prescription No. 6, promptly with 0.5% magnesium stearate and 0.2% micropowder silica gel as fluidizer and lubricant.

(3) selection of aspartame (sweeting agent) consumption

Get 3 parts of the granules of above-mentioned experiment, every part of 5g,, press table 2 and add not commensurability aspartame respectively, add the 200ml hot water dissolving behind the mixing, attempt mouthfeel, record the results are shown in Table 20.

Table 20 aspartame (sweeting agent) consumption experimental result

By table 20 result as seen, mouthfeel is better when adding A Patan 0.9%, determines that the aspartame consumption is 0.9%.

3. adjuvant is originated and quality standard

This product used auxilliary grain source and quality standard see Table 21.

Table 21 auxilliary grain source and quality standard see Table 21

The mensuration of 4 critical relative humiditys

Method: adopt the dry powder method to measure the particulate critical relative humidity of this product.Get 7 small-sized glass exsiccators, press the saturated solution of the different relative humidity salt of table 22 preparation, put into above-mentioned exsiccator respectively, add a cover 25 ℃ of balances of room temperature 24 hours.Other gets 14 weighing botles that size is identical, and precision weighing is put into the about 0.3g of sample, then weighing botle is put into exsiccator, places 24 hours the sample appearance of precision weighing, and record again for 25 ℃.The results are shown in Table 22.

Particulate hygroscopic capacity and cosmetic variation under the various relative humiditys of table 22

Annotate: outward appearance is the brown yellow granule shape before the granule test, good fluidity.

The visible this product granule of table 22 result has certain moisture, and its critical relative humidity is 60%.

The experiment of experimental example 11 assays

1, instrument and reagent

Instrument favour spectrum 1100-type high performance liquid chromatograph.

Reagent water is a pure water, and methanol is that top grade is pure, and other reagent are analytical pure.

Stilbene glucoside reference substance: lot number is provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute: 110844-200404 is the assay specification.

The Radix Polygoni Multiflori Preparata decoction pieces is purchased in Chinese Medicinal Materials Co.

2, chromatographiccondition

Chromatographic column Luna 5 μ m, C ₁₈(2), 250 * 4.60mm100A.

Mobile phase methanol-0.2% phosphoric acid solution (29:71).

30 ℃ of column temperatures.

Detect wavelength 320nm.

Flow velocity 1ml/min.

Separating degree (R) stilbene glucoside peak and adjacent assorted peak separating degree are greater than 1.5.

Number of theoretical plate calculates by the stilbene glucoside peak should be not less than 3000.

3, method and result

(1) mobile phase is selected to select test by mobile phase, thinks that methanol---0.2% phosphoric acid solution water (29:71) is mobile phase, and stilbene glucoside peak and adjacent peak reach baseline separation.

(2) linear relationship is investigated

Accurate stilbene glucoside reference substance solution (0.0998mg/ml) 0.5,1.0,1.5,2.0, the 3mL of drawing puts respectively in the 10mL measuring bottle, adds 50% methanol and is diluted to scale, shake up, get stilbene glucoside reference substance series solution, accurate each reference substance solution 20 μ L that draw inject chromatograph of liquid, measure peak area, with stilbene glucoside sample size (μ g number) is abscissa, and peak area is a vertical coordinate, the drawing standard curve, its regression equation is: Y=3483.99X-4.3784, r=0.9999.The result shows that stilbene glucoside has good linear relationship in 0.0988～0.5928 μ g scope, the results are shown in Table 23.

Table 23 stilbene glucoside linear relationship

(3) extraction solvent is selected

Sample thief number part adds methanol, 50% methanol, 50% ethanol respectively, and supersound process 30 minutes is pressed the text method and measured, and the results are shown in Table 24.

The different solvents of table 24 extract comparative result

The above results shows, adopts above-mentioned 3 kinds of solvents to extract, and stilbene glucoside content is more or less the same, but shallow with the methanol extraction solution colour, is beneficial to protection chromatograph column life, so select methanol as extraction solvent.

(4) extracting method choosing and extraction time are selected

Sample thief number part adds methanol, and respectively with supersound process 10,20,30 minutes, modes such as reflux 30 minutes are extracted, and press the text method to measure, and the results are shown in Table 25

Stilbene glucoside content in the different extracting mode samples of table 25

The above results shows, adopts 2 kinds of modes and different ultrasonic time to extract, and the stilbene glucoside content difference is little, for simplicity, adopts supersound extraction to get final product in 20 minutes.

(5) precision test

(6) stability test

Precision takes by weighing sample, and (lot number: 050511), make need testing solution by the text method, respectively at preparing the back 0,3,6,9,12,24 hour, measure in accordance with the law, the result shows that need testing solution is basicly stable in 24 hours, the results are shown in Table 26.

Table 26 stability test result

(7) replica test

Press the text content assaying method, to same lot number (lot number: 050511) sample, measure, try to achieve relative standard deviation less than 5%, the results are shown in Table 27.

Table 27 replica test

(8) recovery test

Adopt the application of sample absorption method, precision takes by weighing the same lot number (lot number: the about 0.1g of sample 050511) of known content, accurate respectively stilbene glucoside reference substance solution (0.1620mg/ml) 3.0ml that adds, press the preparation method preparation and the above-mentioned chromatographic condition of text need testing solution, measure, be calculated as follows the response rate, the results are shown in Table 28.

Table 28 stilbene glucoside determination of recovery rates result

Following embodiment all can realize the described effect of above-mentioned experimental example

The specific embodiment

Embodiment 1:

Radix Polygoni Multiflori Preparata 600g Herba Epimedii 400g Semen Cuscutae 600g

Rhizoma Polygoni Cuspidati 400g Radix Paeoniae Alba 400g Rhizoma Chuanxiong 400g

Adjuvant: lactose 560g magnesium stearate 5g micropowder silica gel 2g aspartame 9g;

Method for making: above Six-element, Herba Epimedii, Semen Cuscutae, the Radix Paeoniae Alba decoct with water three times, and each 1.5 hours, amount of water was 14 times of medical material amount; Collecting decoction, being concentrated into 50 ℃, relatively to survey density be 1.04-1.05, adds ethanol and make medicinal liquid contain the alcohol amount to reach 50%, and liquid is crossed in 5-10 ℃ of cold preservation, filters, and the precipitate with ethanol supernatant is standby; Radix Polygoni Multiflori Preparata, Rhizoma Polygoni Cuspidati, Rhizoma Chuanxiong add 6 times of amounts of ethanol with 60% alcohol reflux 2 times at every turn, reflux 2 hours, merge alcohol extract, filter, and promptly get alcohol extract; Get alcohol extract and above-mentioned precipitate with ethanol supernatant and merge, reclaim ethanol to there not being the alcohol flavor, be concentrated into relative density about 1.30, subtract and press dry dryly below 70 ℃, dry extract was pulverized 100 mesh sieves; Add lactose, magnesium stearate, micropowder silica gel, aspartame mixing, dry-pressing is granulated, and is packaged into bag, the 5g/ bag; Promptly.

Embodiment 2:

Radix Polygoni Multiflori Preparata 650g Herba Epimedii 350g Semen Cuscutae 650g

Rhizoma Polygoni Cuspidati 350g Radix Paeoniae Alba 550g Rhizoma Chuanxiong 350g;

Adjuvant: lactose 660g magnesium stearate 9g micropowder silica gel 4g aspartame 12g;

Method for making: above Six-element, Herba Epimedii, Semen Cuscutae, the Radix Paeoniae Alba decoct with water three times, and each 2 hours, amount of water was 15 times of medical material amount; Collecting decoction, being concentrated into 55 ℃, relatively to survey density be 1.04-1.05, adds ethanol and make medicinal liquid contain the alcohol amount to reach 55%, and liquid is crossed in 5-10 ℃ of cold preservation, filters, and the precipitate with ethanol supernatant is standby; Radix Polygoni Multiflori Preparata, Rhizoma Polygoni Cuspidati, Rhizoma Chuanxiong add 6 times of amounts of ethanol with 66% alcohol reflux 3 times at every turn, reflux 2 hours, merge alcohol extract, filter, and promptly get alcohol extract; Get alcohol extract and above-mentioned precipitate with ethanol supernatant and merge, reclaim ethanol to there not being the alcohol flavor, be concentrated into relative density about 1.30, subtract and press dry dryly below 60 ℃, dry extract was pulverized 100 mesh sieves; Add lactose, magnesium stearate, micropowder silica gel, aspartame mixing, dry-pressing is granulated, and is packaged into bag, the 5g/ bag; Promptly.

Embodiment 3:

Radix Polygoni Multiflori Preparata 550g Herba Epimedii 580g Semen Cuscutae 500g

Rhizoma Polygoni Cuspidati 460g Radix Paeoniae Alba 360g Rhizoma Chuanxiong 460g;

Method for making: above Six-element, Herba Epimedii, Semen Cuscutae, the Radix Paeoniae Alba decoct with water three times, and each 2 hours, amount of water was 15 times of medical material amount; Collecting decoction, being concentrated into 55 ℃, relatively to survey density be 1.04-1.05, adds ethanol and make medicinal liquid contain the alcohol amount to reach 55%, and liquid is crossed in 5-10 ℃ of cold preservation, filters, and the precipitate with ethanol supernatant is standby; Radix Polygoni Multiflori Preparata, Rhizoma Polygoni Cuspidati, Rhizoma Chuanxiong add 6 times of amounts of ethanol with 66% alcohol reflux 3 times at every turn, reflux 2 hours, merge alcohol extract, filter, and promptly get alcohol extract; Get alcohol extract and above-mentioned precipitate with ethanol supernatant and merge, reclaim ethanol to there not being the alcohol flavor, be concentrated into relative density about 1.30, subtract and press dry dryly below 60 ℃, common process is made tablet.

Detection method:

Differentiate:

A. get present composition tablet, 60 order porphyrizes take by weighing 1/5 unit formulation, add ethanol 20mL, ultrasonic place 20 minutes filters, and filtrate evaporate to dryness, residue add water 10mL dissolving, with ethyl acetate extraction 2 times, each 20mL, combined ethyl acetate liquid, evaporate to dryness, residue adds water 10mL dissolving, crosses 2g, 14-30 order, internal diameter 1.8cm polyamide column, the water 50mL of elder generation eluting discards water liquid, reuse 70% ethanol 50mL eluting, collect the eluent evaporate to dryness, residue adds methanol 2mL dissolving, as need testing solution; Other gets the icariin reference substance, adds methanol and makes the methanol solution that every 1mL contains 0.5mg, in contrast product solution; Test according to thin layer chromatography, draw each 4 μ L of need testing solution and reference substance solution, putting respectively on same silica gel g thin-layer plate, is developing solvent with lower floor's solution of 7: 2.5: 1 chloroform-methanol-water, saturated 15 minutes, launch 9cm, take out, dry, spray is with 1% aluminum chloride alcoholic solution, place after 30 minutes, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with contrast chromatograph relevant position on, show the speckle of same color fluorescence;

B, get present composition tablet, 60 order porphyrizes take by weighing 1/5 unit formulation, add chloroform 20mL, and supersound process 10 minutes filters, and filtrate evaporate to dryness, residue add methanol 1mL makes dissolving, as need testing solution; Other gets Rhizoma Polygoni Cuspidati control medicinal material 60 order 1g, makes the 1mL methanol solution with method, in contrast product medical material solution; According to the thin layer chromatography test, draw need testing solution, each 4 μ L of control medicinal material solution, put respectively on same silica gel g thin-layer plate, with 9: 1: 0.2 toluene-ethyl acetate-formic acid was developing solvent, and presaturation 15 minutes launches 9cm, take out, dry, under visible light, inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical yellow spotting;

C, get present composition tablet, 60 order porphyrizes take by weighing 1/5 unit formulation, the 20mL that adds diethyl ether, and supersound process 20 minutes filters, and filtrate evaporate to dryness, residue add ethyl acetate 5mL makes dissolving, as need testing solution; Other gets Rhizoma Chuanxiong control medicinal material 60 order 0.5g, makes the 5mL ethyl acetate solution with method, in contrast medical material solution; According to the thin layer chromatography test, draw each 8 μ L of need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with 9.5: 0.5 petroleum ether (60-90 ℃)-ethyl acetates was developing solvent, and presaturation 15 minutes launches 9cm, take out, dry, put under the 365nm ultraviolet light and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the bright fluorescence speckle of same color;

D; get present composition tablet, 60 order porphyrizes take by weighing 1/5 unit formulation; add methanol 20mL, and supersound process 10 minutes filters; filtrate evaporate to dryness, residue add water 10mL makes dissolving, 3000 rev/mins of centrifugal 10 clocks; get supernatant and cross HPD100, high 8cm, internal diameter 1cm macroporous resin column; first water 50mL eluting, discards water liquid, reuse 30% ethanol 50mL eluting; collect eluent, and evaporate to dryness, residue add water 10mL dissolving; use ether extraction 3 times, and each 20mL discards ether liquid; water liquid evaporate to dryness, and residue adds the 5mL dissolve with methanol, crosses 2g; internal diameter 1cm neutral alumina post, uses the 20mL methanol-eluted fractions, collects eluent; evaporate to dryness, residue add methanol 1mL makes dissolving, as need testing solution; Other gets the peoniflorin reference substance, adds ethanol and makes the solution that every 1mL contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw need testing solution 10 μ L, reference substance solution 4 μ L, put respectively on same silica gel g thin-layer plate, with 40: 5: 10: the solution of 0.2 chloroform-ethyl acetate-methanol-formic acid was developing solvent, and presaturation 20 minutes launches 9cm, take out, dry, spray is with 5% vanillin sulfuric acid solution, and 100 ℃ (or hair dryer) is heated to the speckle colour developing; In the test sample chromatograph, with reference substance chromatograph relevant position on, show the speckle of same color;

Assay:

Embodiment 4:

Radix Polygoni Multiflori Preparata 650g Herba Epimedii 380g Semen Cuscutae 660g

Rhizoma Polygoni Cuspidati 450g Radix Paeoniae Alba 460g Rhizoma Chuanxiong 460g

Common process is made oral liquid.

Embodiment 5: detection method

The detection method of implementing at the granular preparation of embodiment 1:

Differentiate:

B, get present composition granule, 60 order porphyrizes take by weighing 1/5 unit formulation, the 20mL that adds diethyl ether, and supersound process 20 minutes filters, and filtrate evaporate to dryness, residue add ethyl acetate 5mL makes dissolving, as need testing solution; Other gets Rhizoma Chuanxiong control medicinal material 60 order 0.5g, makes the 5mL ethyl acetate solution with method, in contrast medical material solution; According to the thin layer chromatography test, draw each 8 μ L of need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with petroleum ether (60-90 ℃)-ethyl acetate (9.5: 0.5) is developing solvent, and presaturation 15 minutes launches 9cm, take out, dry, put under the 365nm ultraviolet light and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the bright fluorescence speckle of same color;

Assay:

According to high performance liquid chromatography, chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler; 29: 71 alcohol-0.2% of first phosphoric acid solution is a mobile phase; The detection wavelength is 320nm; Number of theoretical plate is by 2,3,5, and 4 '-tetrahydroxystilbene-2-O-β-D-glucoside peak calculates should be not less than 3000;

The preparation of reference substance solution: precision takes by weighing 2,3, and 5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside reference substance 5mg, put in the 50mL measuring bottle, add 50% dissolve with methanol, and be diluted to scale, shake up, precision is measured 4mL, puts in the 25mL measuring bottle, adds 50% methanol and is diluted to scale, shake up, promptly get every 1mL and contain 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside 16 μ g;

The content under the present composition granule content uniformity item is got in the preparation of need testing solution, and 80 order porphyrizes are got 1/20 unit formulation, and accurate the title decides, put in the tool plug conical flask, the accurate methanol 25mL that adds claims to decide weight, power 100W, frequency 40KHz supersound process 20 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, get subsequent filtrate 4mL, put in the 10mL measuring bottle, thin up is to scale, shake up, filter with 0.5 μ m microporous filter membrane, promptly;

Algoscopy: accurate respectively reference substance solution and each 20 μ L of need testing solution of drawing, inject chromatograph of liquid, measure, promptly;

Present composition granule per unit unit formulation contain Radix Polygoni Multiflori Preparata with 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside (C ₂₀H ₂₂O ₉) meter, must not be less than 12.5mg.

Embodiment 6: detection method

The detection method of implementing at the granular preparation of embodiment 2:

Differentiate:

C, get present composition granule, 60 order porphyrizes take by weighing 1/5 unit formulation, the 20mL that adds diethyl ether, and supersound process 20 minutes filters, and filtrate evaporate to dryness, residue add ethyl acetate 5mL makes dissolving, as need testing solution; Other gets Rhizoma Chuanxiong control medicinal material 60 order 0.5g, makes the 5mL ethyl acetate solution with method, in contrast medical material solution; According to the thin layer chromatography test, draw each 8 μ L of need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with petroleum ether (60-90 ℃)-ethyl acetate (9.5: 0.5) is developing solvent, and presaturation 15 minutes launches 9cm, take out, dry, put under the 365nm ultraviolet light and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the bright fluorescence speckle of same color;

Assay:

Present composition granule per unit preparation contain Radix Polygoni Multiflori Preparata with 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside (C ₂₀H ₂₂O ₉) meter, must not be less than 12.5mg.

Embodiment 7: detection method

Differentiate:

A. get present composition granule, 60 order porphyrizes take by weighing 1/5 unit formulation, add ethanol 20mL, supersound process 20 minutes filters, and filtrate evaporate to dryness, residue add water 10mL dissolving, with ethyl acetate extraction 2 times, each 20mL, combined ethyl acetate liquid, evaporate to dryness, residue adds water 10mL dissolving, crosses 2g, 14-30 order, internal diameter 1.8cm polyamide column, the water 50mL of elder generation eluting discards water liquid, reuse 70% ethanol 50mL eluting, collect the eluent evaporate to dryness, residue adds methanol 2mL dissolving, as need testing solution; Other gets the icariin reference substance, adds methanol and makes the methanol solution that every 1mL contains 0.5mg, in contrast product solution; Test according to thin layer chromatography, draw each 4 μ L of need testing solution and reference substance solution, putting respectively on same silica gel g thin-layer plate, is developing solvent with lower floor's solution of 7:2.5:1 chloroform-methanol-water, saturated 15 minutes, launch 9cm, take out, dry, spray is with 1% aluminum chloride alcoholic solution, place after 30 minutes, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with contrast chromatograph relevant position on, show the speckle of same color fluorescence;

B, get present composition granule, 600 order porphyrizes take by weighing 1/5 unit formulation, add chloroform 20mL, and supersound process 10 minutes filters, and filtrate evaporate to dryness, residue add methanol 1mL makes dissolving, as need testing solution; Other gets Rhizoma Polygoni Cuspidati control medicinal material 60 order 1g, makes the 1mL methanol solution with method, in contrast product medical material solution; According to the thin layer chromatography test, draw need testing solution, each 4 μ L of control medicinal material solution, put respectively on same silica gel g thin-layer plate, with 9:1:0.2 toluene-ethyl acetate-formic acid is developing solvent, and presaturation 15 minutes launches 9cm, take out, dry, under visible light, inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical yellow spotting;

C, get present composition granule, 60 order porphyrizes take by weighing 1/5 unit formulation, the 20mL that adds diethyl ether, and supersound process 20 minutes filters, and filtrate evaporate to dryness, residue add ethyl acetate 5mL makes dissolving, as need testing solution; Other gets Rhizoma Chuanxiong control medicinal material 60 order 0.5g, makes the 5mL ethyl acetate solution with method, in contrast medical material solution; According to the thin layer chromatography test, draw each 8 μ L of need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with petroleum ether (60-90 ℃)-ethyl acetate (9.5:0.5) is developing solvent, and presaturation 15 minutes launches 9cm, take out, dry, put under the 365nm ultraviolet light and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the bright fluorescence speckle of same color;

D; get present composition granule, 60 order porphyrizes take by weighing 1/5 unit formulation; add methanol 20mL, and supersound process 10 minutes filters; filtrate evaporate to dryness, residue add water 10mL makes dissolving, 3000 rev/mins of centrifugal 10 clocks; get supernatant and cross HPD100, high 8cm, internal diameter 1cm macroporous resin column; first water 50mL eluting, discards water liquid, reuse 30% ethanol 50mL eluting; collect eluent, and evaporate to dryness, residue add water 10mL dissolving; use ether extraction 3 times, and each 20mL discards ether liquid; water liquid evaporate to dryness, and residue adds the 5mL dissolve with methanol, crosses 2g; internal diameter 1cm neutral alumina post, uses the 20mL methanol-eluted fractions, collects eluent; evaporate to dryness, residue add methanol 1mL makes dissolving, as need testing solution; Other gets the peoniflorin reference substance, adds ethanol and makes the solution that every 1mL contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw need testing solution 10 μ L, reference substance solution 4 μ L, put respectively on same silica gel g thin-layer plate, solution with 40:5:10:0.2 chloroform-ethyl acetate-methanol-formic acid is developing solvent, and presaturation 20 minutes launches 9cm, take out, dry, spray is with 5% vanillin sulfuric acid solution, and 100 ℃ (or hair dryer) is heated to the speckle colour developing; In the test sample chromatograph, with reference substance chromatograph relevant position on, show the speckle of same color;

Assay:

According to high performance liquid chromatography, chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler; First 29:71 alcohol-0.2% phosphoric acid solution is a mobile phase; The detection wavelength is 320nm; Number of theoretical plate is by 2,3,5, and 4 '-tetrahydroxystilbene-2-O-β-D-glucoside peak calculates should be not less than 3000;

The preparation of need testing solution: get the content under the present composition granule content uniformity item, 80 order porphyrizes are got 1/20 unit formulation, and accurate the title decides, put in the tool plug conical flask, the accurate methanol 25mL that adds claims to decide weight, power 100W, frequency 40KHz supersound process 20 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, get subsequent filtrate 4mL, put in the 10mL measuring bottle, thin up is to scale, shake up, filter with 0.5 μ m microporous filter membrane, promptly;

Algoscopy: accurate respectively reference substance solution and each 20 μ L of need testing solution of drawing, inject chromatograph of liquid, measure, promptly; Present composition granule per unit preparation contain Radix Polygoni Multiflori Preparata with 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside (C ₂₀H ₂₂O ₉) meter, must not be less than 12.5mg.

Claims

1. pharmaceutical composition for the treatment of climacteric syndrome is characterized in that the crude drug of this pharmaceutical composition consists of:

2. pharmaceutical composition as claimed in claim 1 is characterized in that the crude drug of this pharmaceutical composition consists of:

3. pharmaceutical composition as claimed in claim 2 is characterized in that the crude drug of this pharmaceutical composition consists of:

The Radix Paeoniae Alba 400 weight portion Rhizoma Chuanxiongs 400 weight portions.

4. pharmaceutical composition as claimed in claim 2 is characterized in that the crude drug of this pharmaceutical composition consists of:

5. as described any one pharmaceutical composition of claim 1-4, it is characterized in that adding in this pharmaceutical composition following adjuvant and make granule: lactose 400-700 weight portion magnesium stearate 3-10 weight portion micropowder silica gel 1-6 weight portion aspartame 3-15 weight portion.

6. pharmaceutical composition as claimed in claim 5 is characterized in that adding following adjuvant and makes granule:

Lactose 700 weight portion magnesium stearate 5 weight portion micropowder silica gels 5 weight portion aspartames 6 weight portions.

7. pharmaceutical composition as claimed in claim 5 is characterized in that adding following adjuvant and makes granule:

Lactose 560 weight portion magnesium stearate 5 weight portion micropowder silica gels 2 weight portion aspartames 9 weight portions.

8. preparation of drug combination method as claimed in claim 5 is characterized in that this method is:

Herba Epimedii, Semen Cuscutae, the Radix Paeoniae Alba decoct with water 2-4 time, and each 1-2 hour, amount of water was 12-16 a times of medical material amount; Collecting decoction, being concentrated into 40-60 ℃, to survey relative density be 1.04-1.05, adds ethanol and make medicinal liquid contain the alcohol amount to reach 40-60%, and liquid is crossed in 3-10 ℃ of cold preservation, filters, and the precipitate with ethanol supernatant is standby; The alcohol reflux of Radix Polygoni Multiflori Preparata, Rhizoma Polygoni Cuspidati, Rhizoma Chuanxiong usefulness 50-70% 2-3 time adds ethanol 5-7 at every turn and doubly measures, and backflow 1-3 hour, merge alcohol extract, filter, promptly get alcohol extract; Get alcohol extract and above-mentioned precipitate with ethanol supernatant and merge, reclaim ethanol to there not being the alcohol flavor, be concentrated into relative density 1.20-1.4,80-50 ℃ press dry dryly, and dry extract was pulverized 100 mesh sieves; Add lactose, magnesium stearate, micropowder silica gel, aspartame mixing, dry-pressing is granulated, promptly.

9. preparation of drug combination method as claimed in claim 8 is characterized in that this method is:

Herba Epimedii, Semen Cuscutae, the Radix Paeoniae Alba decoct with water three times, and each 1.5 hours, amount of water was 14 times of medical material amount; Collecting decoction, being concentrated into 50 ℃, relatively to survey density be 1.04-1.05, adds ethanol and make medicinal liquid contain the alcohol amount to reach 50%, and liquid is crossed in 5-10 ℃ of cold preservation, filters, and the precipitate with ethanol supernatant is standby; Radix Polygoni Multiflori Preparata, Rhizoma Polygoni Cuspidati, Rhizoma Chuanxiong add 6 times of amounts of ethanol with 60% alcohol reflux 2 times at every turn, reflux 2 hours, merge alcohol extract, filter, and promptly get alcohol extract; Get alcohol extract and above-mentioned precipitate with ethanol supernatant and merge, reclaim ethanol, be concentrated into relative density 1.30 to there not being the alcohol flavor, 70-60 ℃ of drying under reduced pressure, dry extract was pulverized 100 mesh sieves; Add lactose, magnesium stearate, micropowder silica gel, aspartame mixing, dry-pressing is granulated promptly.

10. as claim 6 or 7 described preparation of drug combination methods, it is characterized in that this method is:

11. preparation of drug combination method as claimed in claim 10 is characterized in that this method is:

12., it is characterized in that this detection method comprises following discrimination method and/or assay as the detection method of claim 1,2,3,4,6 or 7 described pharmaceutical compositions:

Differentiate:

Assay:

The preparation of need testing solution: get the content under the described composite preparation content uniformity item, 70-90 order porphyrize is got the 1/30-1/10 unit formulation, the accurate title, decide, and puts in the tool plug conical flask, and the accurate methanol 25mL that adds claims to decide weight, supersound process 15-30 minute, put coldly, claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, get subsequent filtrate 4mL, put in the 10mL measuring bottle, thin up is to scale, shake up, filter with 0.5 μ m microporous filter membrane, promptly; Algoscopy: respectively accurate each the 20 μ L of reference substance solution and need testing solution that draw, inject chromatograph of liquid, mensuration, that is, and contain Radix Polygoni Multiflori Preparata with 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside C ₂₀H ₂₂O ₉Meter must not be less than the 10-15mg/ unit formulation.

13. the detection method of pharmaceutical composition as claimed in claim 5 is characterized in that this detection method comprises following discrimination method and/or assay:

Differentiate:

A. get described composition granule, 50-70 order porphyrize takes by weighing the 1/10-2/5 unit formulation, adds ethanol 20mL, supersound process 15-30 minute, filter, filtrate evaporate to dryness, residue add water 10mL dissolving, with ethyl acetate extraction 2-3 time, each 20mL, combined ethyl acetate liquid, evaporate to dryness, residue add water 10mL dissolving, cross polyamide column, the water 50mL of elder generation eluting discards water liquid, reuse 60-75% ethanol 50mL eluting, collect the eluent evaporate to dryness, residue adds methanol 2mL dissolving, as need testing solution; Other gets the icariin reference substance, adds methanol and makes the methanol solution that every 1mL contains 0.5mg, in contrast product solution; Test according to thin layer chromatography, draw each 4 μ L of need testing solution and reference substance solution, put respectively on same silica gel g thin-layer plate, with 6-8: 1-3: lower floor's solution of 1-2 chloroform-methanol-water is developing solvent, saturated 10-20 minute, launch 9cm, take out, dry, spray is with 1% aluminum chloride alcoholic solution, place after 20-40 minute, put under the ultra-violet lamp and inspect; In the test sample chromatograph, with contrast chromatograph relevant position on, show the speckle of same color fluorescence;

B, get described composition granule, 50-70 order porphyrize takes by weighing the 1/10-2/5 unit formulation, adds chloroform 20mL, and supersound process 5-15 minute, filter, filtrate evaporate to dryness, residue add methanol 1mL makes dissolving, as need testing solution; Other gets Rhizoma Polygoni Cuspidati control medicinal material 50-70 order 1g, adds chloroform 20mL, and supersound process 5-15 minute, filter, filtrate evaporate to dryness, residue add methanol 1mL makes dissolving, makes the 1mL methanol solution, in contrast product medical material solution; According to the thin layer chromatography test, draw need testing solution, each 4 μ L of control medicinal material solution, put respectively on same silica gel g thin-layer plate, with 8-11: 1-2: 0.1-0.3 toluene-ethyl acetate-formic acid is developing solvent, presaturation 10-20 minute, launches 9cm, take out, dry, under visible light, inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical yellow spotting;

C, get described composition granule, 50-70 order porphyrize takes by weighing the 1/10-2/5 unit formulation, the 20mL that adds diethyl ether, and supersound process 15-25 minute, filter, filtrate evaporate to dryness, residue add ethyl acetate 5mL makes dissolving, as need testing solution; Other gets Rhizoma Chuanxiong control medicinal material 50-70 order 0.5g, the 20mL that adds diethyl ether, and supersound process 15-25 minute, filter, filtrate evaporate to dryness, residue add ethyl acetate 5mL makes dissolving make 5mL ethyl acetate solution, medical material solution in contrast; Test according to thin layer chromatography, draw each 8 μ L of need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, with 8-11: the 60-90 of 0.4-0.7 ratio ℃ petroleum ether-ethyl acetate is developing solvent, presaturation 10-20 minute, launch 9cm, take out, dry, put under the 365nm ultraviolet light and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the bright fluorescence speckle of same color;

D, get described composition granule, 50-70 order porphyrize takes by weighing the 1/10-2/5 unit formulation, add methanol 20mL, supersound process 5-15 minute, filter, filtrate evaporate to dryness, residue add water 10mL makes dissolving, 3000 rev/mins of centrifugal 5-15 clocks, get supernatant and cross macroporous resin column, first water 50mL eluting discards water liquid, reuse 25-35% ethanol 50mL eluting is collected eluent, evaporate to dryness, residue adds water 10mL dissolving, uses ether extraction 2-4 time, each 20mL, discard ether liquid, water liquid evaporate to dryness, residue adds the 5mL dissolve with methanol, cross the neutral alumina post, use the 20mL methanol-eluted fractions, collect eluent, evaporate to dryness, residue adds methanol 1mL makes dissolving, as need testing solution; Other gets the peoniflorin reference substance, adds ethanol and makes the solution that every 1mL contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw need testing solution 10 μ L, reference substance solution 4 μ L, put respectively on same silica gel g thin-layer plate, with 35-45: 4-7: 8-12: the solution of 0.1-0.8 chloroform-ethyl acetate-methanol-formic acid is developing solvent, presaturation 10-25 minute, launches 9cm, take out, dry, spray is with 5% vanillin sulfuric acid solution, and 100 ℃ are heated to the speckle colour developing; In the test sample chromatograph, with reference substance chromatograph relevant position on, show the speckle of same color;

Assay:

The preparation of reference substance solution: precision takes by weighing 2,3, and 5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside reference substance 5mg, put in the 50mL measuring bottle, add the 40-60% dissolve with methanol, and be diluted to scale, shake up, precision is measured 4mL, puts in the 25mL measuring bottle, adds 40-60% methanol and is diluted to scale, shake up, promptly get every 1mL and contain 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside 16 μ g; The preparation of need testing solution: get described composition granule, 70-90 order porphyrize is got the 1/30-1/10 unit formulation, and accurate the title decides, put in the tool plug conical flask, the accurate methanol 25mL that adds claims to decide weight, supersound process 15-30 minute, put coldly, claim to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, get subsequent filtrate 4mL, put in the 10mL measuring bottle, thin up is to scale, shake up, filter with 0.5 μ m microporous filter membrane, promptly; Algoscopy: respectively accurate each the 20 μ L of reference substance solution and need testing solution that draw, inject chromatograph of liquid, mensuration, that is, and contain Radix Polygoni Multiflori Preparata with 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside C ₂₀H ₂₂O ₉Meter must not be less than the 10-15mg/ unit formulation.

14. the detection method of pharmaceutical composition as claimed in claim 12 is characterized in that this detection method comprises the steps:

Differentiate:

A. get described composition granule, 60 order porphyrizes take by weighing 1/5 unit formulation, add ethanol 20mL, supersound process 20 minutes filters, and filtrate evaporate to dryness, residue add water 10mL dissolving, with ethyl acetate extraction 2 times, each 20mL, combined ethyl acetate liquid, evaporate to dryness, residue adds water 10mL dissolving, crosses 2g, 14-30 order, internal diameter 1.8cm polyamide column, the water 50mL of elder generation eluting discards water liquid, reuse 70% ethanol 50mL eluting, collect the eluent evaporate to dryness, residue adds methanol 2mL dissolving, as need testing solution; Other gets the icariin reference substance, adds methanol and makes the methanol solution that every 1mL contains 0.5mg, in contrast product solution; Test according to thin layer chromatography, draw each 4 μ L of need testing solution and reference substance solution, putting respectively on same silica gel g thin-layer plate, is developing solvent with lower floor's solution of 7: 2.5: 1 chloroform-methanol-water, saturated 15 minutes, launch 9cm, take out, dry, spray is with 1% aluminum chloride alcoholic solution, place after 30 minutes, put under the 365nm ultra-violet lamp and inspect; In the test sample chromatograph, with contrast chromatograph relevant position on, show the speckle of same color fluorescence;

B, get described composition granule, 600 order porphyrizes take by weighing 1/5 unit formulation, add chloroform 20mL, and supersound process 10 minutes filters, and filtrate evaporate to dryness, residue add methanol 1mL makes dissolving, as need testing solution; Other gets Rhizoma Polygoni Cuspidati control medicinal material 60 order 1g, adds chloroform 20mL, and supersound process 10 minutes filters, and filtrate evaporate to dryness, residue add methanol 1mL makes dissolving, makes the 1mL methanol solution, in contrast product medical material solution; According to the thin layer chromatography test, draw need testing solution, each 4 μ L of control medicinal material solution, put respectively on same silica gel g thin-layer plate, with 9: 1: 0.2 toluene-ethyl acetate-formic acid was developing solvent, and presaturation 15 minutes launches 9cm, take out, dry, under visible light, inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show identical yellow spotting;

C, get described composition granule, 60 order porphyrizes take by weighing 1/5 unit formulation, the 20mL that adds diethyl ether, and supersound process 20 minutes filters, and filtrate evaporate to dryness, residue add ethyl acetate 5mL makes dissolving, as need testing solution; Other gets Rhizoma Chuanxiong control medicinal material 60 order 0.5g, the 20mL that adds diethyl ether, and supersound process 20 minutes filters, and filtrate evaporate to dryness, residue add ethyl acetate 5mL makes dissolving, makes the 5mL ethyl acetate solution, in contrast medical material solution; Test according to thin layer chromatography, draw each 8 μ L of need testing solution and control medicinal material solution, put respectively on same silica gel g thin-layer plate, 60-90 ℃ of petroleum ether-ethyl acetate with 9.5: 0.5 ratios is developing solvent, presaturation 15 minutes launches 9cm, takes out, dry, put under the 365nm ultraviolet light and inspect; In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the bright fluorescence speckle of same color;

D, get described composition granule, 60 order porphyrizes take by weighing 1/5 unit formulation, add methanol 20mL, supersound process 10 minutes filters, filtrate evaporate to dryness, residue add water 10mL makes dissolving, 3000 rev/mins of centrifugal 10 clocks, get supernatant and cross HPD100, high 8cm, internal diameter 1cm macroporous resin column, the water 50mL of elder generation eluting discards water liquid, reuse 30% ethanol 50mL eluting, collect eluent, evaporate to dryness, residue add water 10mL dissolving, with ether extraction 3 times, each 20mL discards ether liquid, water liquid evaporate to dryness, residue adds the 5mL dissolve with methanol, crosses 2g, internal diameter 1cm neutral alumina post is used the 20mL methanol-eluted fractions, collects eluent, evaporate to dryness, residue add methanol 1mL makes dissolving, as need testing solution; Other gets the peoniflorin reference substance, adds ethanol and makes the solution that every 1mL contains 1mg, in contrast product solution; Test according to thin layer chromatography, draw need testing solution 10 μ L, reference substance solution 4 μ L, put respectively on same silica gel g thin-layer plate, with 40: 5: 10: the solution of 0.2 chloroform-ethyl acetate-methanol-formic acid was developing solvent, and presaturation 20 minutes launches 9cm, take out, dry, spray is with 5% vanillin sulfuric acid solution, and 100 ℃ are heated to the speckle colour developing; In the test sample chromatograph, with reference substance chromatograph relevant position on, show the speckle of same color;

Assay:

According to high performance liquid chromatography, chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler; 29: 71 alcohol-0.2% of first phosphoric acid solution is a mobile phase; The detection wavelength is 320nm; Number of theoretical plate is by 2,3,5, and 4 '-tetrahydroxystilbene-2-O-β-D-glucoside peak calculates should be not less than 3000; The preparation of reference substance solution: precision takes by weighing 2,3, and 5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside reference substance 5mg, put in the 50mL measuring bottle, add 50% dissolve with methanol, and be diluted to scale, shake up, precision is measured 4mL, puts in the 25mL measuring bottle, adds 50% methanol and is diluted to scale, shake up, promptly get every 1mL and contain 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside 16 μ g; The preparation of need testing solution: get the content under the described composition granule content uniformity item, 80 order porphyrizes are got 1/20 unit formulation, and accurate the title decides, put in the tool plug conical flask, the accurate methanol 25mL that adds claims to decide weight, power 100W, frequency 40KHz supersound process 20 minutes is put coldly, claims to decide weight again, supply the weight that subtracts mistake with methanol, shake up, filter, get subsequent filtrate 4mL, put in the 10mL measuring bottle, thin up is to scale, shake up, filter with 0.5 μ m microporous filter membrane, promptly; Algoscopy: respectively accurate each the 20 μ L of reference substance solution and need testing solution that draw, inject chromatograph of liquid, mensuration, that is, and contain Radix Polygoni Multiflori Preparata with 2,3,5,4 '-tetrahydroxystilbene-2-O-β-D-glucoside C ₂₀H ₂₂O ₉Meter must not be less than the 12.5mg/ unit formulation.