CN107884489A

CN107884489A - A kind of detection method of Zaizao Pill

Info

Publication number: CN107884489A
Application number: CN201711083060.4A
Authority: CN
Inventors: 刘莉; 王海宁; 张仕林; 王海洋; 尚秘; 杨飞
Original assignee: Dechangxiang Medical Industry Co Ltd Guiyang
Current assignee: Dechangxiang Medical Industry Co Ltd Guiyang
Priority date: 2017-11-07
Filing date: 2017-11-07
Publication date: 2018-04-06

Abstract

The invention discloses a kind of detection method of Zaizao Pill, the medicine is by prepared RADIX ANGELICAE SINENSIS with yellow rice wine, prepared RHIZOMA CYPERI with vinegar, the root of herbaceous peony, prepared rhizome of rehmannia, donkey-hide gelatin, Poria cocos, Radix Codonopsis, the Radix Astragali, Chinese yam, the bighead atractylodes rhizome, the wine fruit of glossy privet, vinegar processs tortoise plastron, Fructus Corni, teasel root, stir-baked CORTEX EUCOMMIAE with salt solution, saline cistanche, raspberry, cornu cerve degelatinatum, Ligusticum wallichii, the red sage root, the root of bidentate achyranthes, motherwort, corydalis tuber, short pseudo-ginseng, vinegar processs folium artemisiae argyi, fennel seeds, Jehol Ligusticum Rhizome, cuttlebone, wine processs garden burnet, intelligence development, rhizoma alismatis, vinegar processs lotus leaf, bark of ash, the root bark of Chinese wolf-berry, radix cynanchi atrati, bark of tree of heaven, amber, stir-baked RADIX SCUTELLARIAE with yellow rice wine, spina date seed, RADIX POLYGALAE PREPARATA, dried orange peel and radix glycyrrhizae are by being prepared.The detection method includes content assaying method；The content assaying method includes the assay to albiflorin, scutelloside and/or tanshin polyphenolic acid B.

Description

A kind of detection method of Zaizao Pill

Technical field

The present invention relates to a kind of detection method of Zaizao Pill, belong to the field of medicine technology；

Background technology

Gynecological disease, the disease of female reproductive system is refered in particular to, be women common disease, frequently-occurring disease.But because many people are to woman Section's disease lacks due understanding, lacks the health care to body, in addition various bad life habits etc., physiological health is cured per condition Under, cause some women's diseases to be burdened with, and obstinate, bring great inconvenience to normal life, work.

Zaizao Pill records to collect in national standard for traditional Chinese medicines, and Chinese patent drug provincial standard rises national standard part, mark Quasi- numbering：WS-10051 (ZD-0051) -2002, original project is in standard：Character；Microscopical characters；α-cyperolone, Paeoniflorin, Scutelloside, aurantiamarin, the red sage root, Radix Astragali thin layer differentiate；Paeoniflorin content determines.Zaizao Pill is Traditional Chinese medicine historical preparation, by working as Return, rhizoma cyperi, the root of herbaceous peony, prepared rhizome of rehmannia, donkey-hide gelatin, Poria cocos, Radix Codonopsis, the Radix Astragali, Chinese yam, the bighead atractylodes rhizome, the fruit of glossy privet, tortoise version, Fructus Corni, teasel root, Du The secondary taste Chinese medicine of grade 42 composition, it is the compound preparation of one big prescription.Its medicinal material is more, complicated components, only to determine Chinese herbaceous peony in the root of herbaceous peony Medicine glycosides quantifies as index components, makes proper mass standard not perfect enough, it is difficult to controls, evaluates medicine entirety inherent quality.

Therefore, during existing Zaizao Pill assay, only quantified, made as index components using determining Paeoniflorin in the root of herbaceous peony Proper mass standard is not perfect enough, it is difficult to controls, evaluates medicine entirety inherent quality.

The content of the invention

It is an object of the present invention to provide a kind of detection method of Zaizao Pill；The detection method increase is containing measurement Determine item, that is, determine the amount of scutelloside in radix scutellariae medicinal materials；The amount of tanshin polyphenolic acid B in red rooted salvia is determined, detection is accurate, high sensitivity, It is reproducible, reliable results, it can effectively control the quality of the product, it is ensured that the clinical drug effect of medicine.

In order to solve the above technical problems, the present invention adopts the following technical scheme that realization：A kind of detection side of Zaizao Pill Method, the medicine is by prepared RADIX ANGELICAE SINENSIS with yellow rice wine 60-70 parts, prepared RHIZOMA CYPERI with vinegar 60-70 parts, root of herbaceous peony 42-45 parts, prepared rhizome of rehmannia 20-23 parts, donkey-hide gelatin 9-12 Part, Poria cocos 60-70 parts, Radix Codonopsis 20-23 parts, Radix Astragali 20-23 parts, Chinese yam 30-35 parts, bighead atractylodes rhizome 15-17 parts, wine fruit of glossy privet 42-45 Part, vinegar processs tortoise plastron 30-35 parts, Fructus Corni 20-23 parts, teasel root 20-23 parts, stir-baked CORTEX EUCOMMIAE with salt solution 20-23 parts, saline cistanche 9-12 parts, covers basin Sub- 15-17 parts, cornu cerve degelatinatum 3-7 parts, Ligusticum wallichii 42-45 parts, red sage root 20-23 parts, root of bidentate achyranthes 15-17 parts, motherwort 20-23 parts, prolong recklessly Rope 15-17 parts, short pseudo-ginseng 3-7 parts, vinegar process folium artemisiae argyi 42-45 parts, fennel seeds 20-23 parts, Jehol Ligusticum Rhizome 20-23 parts, cuttlebone 30-35 Part, wine processs garden burnet 30-35 parts, intelligence development 9-12 parts, rhizoma alismatis 20-23 parts, vinegar process lotus leaf 15-17 parts, bark of ash 20-23 parts, the root bark of Chinese wolf-berry 20-23 parts, radix cynanchi atrati 42-45 parts, bark of tree of heaven 30-35 parts, amber 3-7 parts, stir-baked RADIX SCUTELLARIAE with yellow rice wine 30-35 parts, spina date seed 9-12 parts, RADIX POLYGALAE PREPARATA 15-17 parts, dried orange peel 30-35 parts and radix glycyrrhizae 20-23 parts are prepared by the following method：The taste medicine of the above 42, the Radix Astragali, salt Du Secondary, prepared rhizome of rehmannia, teasel root, bark of ash, saline cistanche, the root of bidentate achyranthes, the root bark of Chinese wolf-berry add water to cook 2 times, 2 hours every time, collecting decoction, filter, filter Liquid concentrates；Separately take donkey-hide gelatin to add suitable quantity of water molten to merge with above-mentioned concentrate, be concentrated into relative density at 78-82 DEG C be 1.10 it is clear Cream；The taste of remaining Radix Angelicae Sinensis etc. 33 is ground into fine powder, sieves, and mixes；With the general ball of above-mentioned clear cream, condensed pill 1000g, 60- is made 80 DEG C of dryings, sugar coating, polishing, are made pill；The detection method includes content assaying method；The content assaying method bag Include the assay to albiflorin, scutelloside and/or tanshin polyphenolic acid B.

The detection method of foregoing Zaizao Pill, the detection method of content of the albiflorin are：It is high according to Chinese Pharmacopoeia annex Effect liquid phase chromatogram method is tested：

Chromatographic condition and system suitability：Using cyano group bonded silica gel as filler；With methanol-water (11：86-92) it is Mobile phase；Detection wavelength is 230nm；Theoretical cam curve is calculated by Paeoniflorin peak, should be not less than 4000；

The preparation of reference substance solution：Paeoniflorin reference substance 8-12mg is taken, it is accurately weighed, put in 20mL measuring bottles, add methanol molten Solve and be diluted to scale, shake up, precision measures 0.8-1.2mL, puts in 20mL measuring bottles, adds methanol to shake up, produce every to scale 1mL 23-27ug containing Chinese herbaceous peony reference substance solution；

The preparation of need testing solution：Take this product appropriate, remove sugar-coat, it is finely ground, 0.8-1.2g is taken, it is accurately weighed, put tool plug In conical flask, precision adds 45-55% methanol solutions 23-27mL, close plug, weighed weight, is ultrasonically treated, lets cool, then weighed heavy Amount, the weight of less loss is supplied with 45-55% methanol solutions, is shaken up, and is filtered, and precision measures subsequent filtrate 10mL, puts water-bath Back stroke extremely 0.4-0.6mL, again with methanol dissolving, is moved in 10mL measuring bottles, adds methanol to shake up to scale, filter, take subsequent filtrate, produce；

Determination method：Accurate absorption reference substance solution and each 10ul of need testing solution respectively, injection liquid chromatograph, measure, Produce.

Chromatographic condition and system suitability：Using cyano group bonded silica gel as filler；With methanol-water (11：89) it is stream Dynamic phase；Detection wavelength is 230nm；Theoretical cam curve is calculated by Paeoniflorin peak, should be not less than 4000；

The preparation of reference substance solution：Paeoniflorin reference substance 10mg is taken, it is accurately weighed, put in 20mL measuring bottles, add methanol to dissolve And scale is diluted to, shake up, precision measures 1mL, puts in 20mL measuring bottles, adds methanol to shake up to scale, produce every 1mL and contain Chinese herbaceous peony 25ug reference substance solution；

The preparation of need testing solution：Zaizao Pill is taken, removes sugar-coat, it is finely ground, 1g is taken, it is accurately weighed, put tool plug taper In bottle, precision adds 50% methanol solution 25mL, close plug, weighed weight, is ultrasonically treated, lets cool, then weighed weight, with 50% first Alcoholic solution supplies the weight of less loss, shakes up, and filtration, precision measures subsequent filtrate 10mL, puts water-bath Back stroke to 0.5mL, again with methanol Dissolving, is moved in 10mL measuring bottles, adds methanol to shake up to scale, filter, take subsequent filtrate, produce；

The detection method of foregoing Zaizao Pill, the supersound process are；It is 245-255W, frequency 33- in power 0.8-1.2h is ultrasonically treated under 37kHz.

The detection method of foregoing Zaizao Pill, the detection method of content of the scutelloside are：It is high according to Chinese Pharmacopoeia annex Effect liquid phase chromatogram method is tested：

Chromatographic condition and system suitability test：Using octadecylsilane chemically bonded silica as filler；With methanol -0.2% Phosphoric acid solution (47:50-56) it is mobile phase：Detection wavelength is 275-285nm；Theoretical cam curve is calculated by scutelloside peak, should not Less than 2500；

The preparation of reference substance solution：Precision claims scutelloside reference substance appropriate, adds 55-65% ethanol that every 1ml is made and contains 48-52 μ g solution, shakes up, and produces；

The preparation of need testing solution：Zaizao Pill is taken, removes sugar-coat, it is finely ground, 0.5g is taken, it is accurately weighed, put tool plug cone In shape bottle, precision adds 55-65% ethanol solutions 23-27mL, close plug, weighed weight, is ultrasonically treated, lets cool, then weighed weight, The weight of less loss is supplied with 55-65% ethanol solutions, is shaken up, is filtered, is taken subsequent filtrate, produce；

Chromatographic condition and system suitability test：Using octadecylsilane chemically bonded silica as filler；With methanol -0.2% Phosphoric acid solution (47:53) it is mobile phase：Detection wavelength is 280m；Theoretical cam curve is calculated by scutelloside peak, should be not less than 2500；

The preparation of reference substance solution：Precision claims scutelloside reference substance appropriate, adds 60% ethanol that every 1ml is made molten containing 50 μ g Liquid, shake up, produce；

The preparation of need testing solution：Zaizao Pill is taken, removes sugar-coat, it is finely ground, 0.5g is taken, it is accurately weighed, put tool plug cone In shape bottle, precision adds 60% ethanol solution 25mL, close plug, weighed weight, is ultrasonically treated, lets cool, then weighed weight, with 60% Ethanol solution supplies the weight of less loss, shakes up, and filtration, takes subsequent filtrate, produces：

The detection method of foregoing Zaizao Pill, the supersound process are；It is 245-255W, frequency 30- in power 40-50 minutes are ultrasonically treated under 40kHz.

The detection method of foregoing Zaizao Pill, the detection method of content of the tanshin polyphenolic acid B are：It is high according to Chinese Pharmacopoeia annex Effect liquid phase chromatogram method is tested：

Chromatographic condition and system suitability test：Using octadecylsilane chemically bonded silica as filler；With methanol -0.1% Phosphoric acid solution (40：55-65) it is mobile phase；Detection wavelength is 285-287nm；Theoretical cam curve is calculated by tanshin polyphenolic acid B peak, should not Less than 2000；

The preparation of reference substance solution：Precision claims tanshin polyphenolic acid B reference substance, adds 75-85% methanol that every 1ml μ containing 38-42 g are made Solution, shake up, produce；

The preparation of need testing solution：Zaizao Pill is taken, removes sugar-coat, it is finely ground, 1g is taken, it is accurately weighed, put tool plug taper In bottle, precision adds 75-85% methanol solutions 20-30mL, close plug, weighed weight, is heated to reflux 25-35 minutes, lets cool, then claims Determine weight, the weight of less loss is supplied with 78-82% methanol solutions, is shaken up, filter, take subsequent filtrate, produce；

Chromatographic condition and system suitability test：Using octadecylsilane chemically bonded silica as filler；With methanol -0.1% Phosphoric acid solution (40：60) it is mobile phase；Detection wavelength is 286nm；Theoretical cam curve is calculated by tanshin polyphenolic acid B peak, should be not less than 2000；

The preparation of reference substance solution：Precision claims tanshin polyphenolic acid B reference substance, adds 80% methanol that the solution that every 1ml contains 40 μ g is made, Shake up, produce；

The preparation of need testing solution：Zaizao Pill is taken, removes sugar-coat, it is finely ground, 1g is taken, it is accurately weighed, put tool plug taper In bottle, precision adds 80% methanol solution 25mL, close plug, weighed weight, is heated to reflux 30 minutes, lets cool, then weighed weight, uses 80% methanol solution supplies the weight of less loss, shakes up, and filtration, takes subsequent filtrate, produces；

The detection method of foregoing Zaizao Pill, the medicine so detect：

Character：This product is sugar-coat condensed pill, removes sugar-coat and shows brown color to sepia；Mildly bitter flavor, slightly fiber crops.

Differentiate：(1) this product is taken, puts micro- Microscopic observation：Irregular branched agglomerate is colourless, meets chloraldurate liquid and dissolves； Hyphae colorless or light brown, diameter(Poria cocos)；Fiber is faint yellow, fusiformis, wall thickness, and hole ditch is thin (radix scutellariae)；It is T-shaped non- Glandular hairs are bent, handleIndividual cell, often come off (folium artemisiae argyi)；

(2) this product 15g is taken, it is finely ground, add petroleum ether (60~90 DEG C) 50ml, be heated to reflux 40 minutes, filter, filtrate is waved Dry, residue adds ethyl acetate 1ml to make dissolving, as need testing solution；α-cyperolone reference substance separately is taken, adds ethyl acetate to be made often Solution of the 1ml containing 1mg, as reference substance solution；Tested according to thin-layered chromatography (one B of annex VI of Chinese Pharmacopoeia version in 2010), Draw need testing solution 10 μ l, the μ l of reference substance solution 2, put respectively on same silica gel g thin-layer plate, make into strips, with hexamethylene- Ethyl acetate (9:1) it is solvent, deploys, take out, dry, sprays with dinitrophenylhydrazine test solution, place 1-5min；Test sample chromatogram In, on position corresponding with reference substance chromatogram, show the orange red spot of identical；

(3) this product 15g is taken, it is finely ground, add methanol 50ml, be heated to reflux 45 minutes, let cool, filter, filtrate is evaporated, and residue adds Water 30ml makes dissolving, with ether shaking extraction 2 times, each 20ml, discards ether liquid, is extracted 3 times with the shaking of water saturated n-butanol, Each 25ml, merge n-butanol liquid, then the washing 3 times with n-butanol saturation, each 20ml, take n-butanol liquid, be evaporated, residue adds Methanol 1ml makes dissolving, adds neutral alumina 2g, is mixed thoroughly in water-bath, dries, loading neutral alumina column (100~200 mesh, 2g, internal diameter 10mm, dry column-packing) on, with acetate-methanol (1:1) 40ml is eluted, and is collected eluent, is evaporated, residue adds second Alcohol 1ml makes dissolving, as need testing solution；Paeoniflorin reference substance separately is taken, adds ethanol that solution of every 1ml containing 1mg is made, as right According to product solution；Tested according to thin-layered chromatography (one B of annex VI of Chinese Pharmacopoeia version in 2010), draw each 10 μ of above two solution L, put respectively on same silica gel g thin-layer plate, with chloroform-acetate-methanol-formic acid (40:5:10:0.2) it is expansion Agent, deploy, take out, dry, spray with 5% vanillin-sulfuric acid solution, it is clear to be heated to spot development at 105 DEG C.Test sample chromatogram In, on position corresponding with reference substance chromatogram, show the spot of same color；

(4) this product 15g is taken, finely ground, add diethyl ether 50ml, is heated to reflux 30 minutes, filtration, discards ether liquid；The dregs of a decoction fling to second Ether, add methanol 50ml, be heated to reflux 45 minutes, filter, filtrate is evaporated, and residue adds water 25ml to make dissolving, filtration, filtrate hydrochloric acid PH value is adjusted to 2, with ethyl acetate shaking extraction 2 times, each 20ml, combined ethyl acetate liquid, is evaporated, residue adds methanol 1ml Make dissolving, as need testing solution；Scutelloside reference substance separately is taken, adds methanol that solution of every 1ml containing 1mg is made, as reference substance Solution；Tested according to thin-layered chromatography (one B of annex VI of Chinese Pharmacopoeia version in 2010), draw each 10 μ l of above two solution, point Other point in same on the silica gel g thin-layer plate of 4% sodium acetate, to make into strips, with ethyl acetate-butanone-formic acid-water (5:3:1: 1) it is solvent, puts presaturation 30 minutes in expansion cylinder, deploy, takes out, dry, spray with 5% ferric trichloride ethanol solution.For examination In product chromatogram, on position corresponding with reference substance chromatogram, show the spot of same color；

(5) aurantiamarin reference substance is taken, adds methanol that saturated solution is made, as reference substance solution；According to thin-layered chromatography (China One B of annex VI of pharmacopeia version in 2010) to test, the need testing solution and above-mentioned reference substance solution under absorption [discriminating] (4) item are each 10 μ l, put respectively in same on the silica gel g thin-layer plate of 1% sodium hydroxide, to make into strips, with chloroform-methanol-water (32: 17:5) lower floor's solution is solvent, puts presaturation 30 minutes in expansion cylinder, is deployed, and takes out, dries；Spray is tried with alchlor Liquid, put and inspected under ultraviolet lamp (365nm)；In test sample chromatogram, on position corresponding with reference substance chromatogram, show same color Fluorescence spot；

(6) 25 g of this product are taken, finely ground, add diethyl ether 80ml, is ultrasonically treated 30 minutes, and filtration, filtrate volatilizes, and residue adds second Acetoacetic ester 2ml makes dissolving, as need testing solution.Red sage root control medicinal material separately is taken, is made in the same way of control medicinal material solution；The red sage root is taken again The A reference substances of ketone II, add ethyl acetate that solution of every 1ml containing 1mg is made, as reference substance solution；According to thin-layered chromatography (middle traditional Chinese medicines One B of annex VI of allusion quotation version in 2010) experiment, the μ l of need testing solution 20, control medicinal material solution and each 5 μ l of reference substance solution are drawn, Put respectively on same silica gel g thin-layer plate, with benzene-ethyl acetate (19:1) it is solvent, deploys, takes out, dry.Test sample color In spectrum, on position corresponding with control medicinal material and reference substance chromatogram, show the spot of same color；

(7) this product 25g is taken, it is finely ground, add water saturated n-butanol 80ml, be ultrasonically treated 30 minutes, filtration, filtrate ammonification examination Liquid shaking extraction 2 times, each 25ml, discards ammoniacal liquor, and n-butanol liquid with the water washing 2 times of n-butanol saturation, each 25ml, is abandoned again Aqueous is gone, n-butanol liquid is evaporated, and residue adds methanol 2ml to make dissolving, as need testing solution；Separately take Radix Astragali control medicinal material, same to method Control medicinal material solution is made；Astragaloside IV reference substance is taken again, adds methanol that solution of every 1ml containing 1mg is made, it is molten as reference substance Liquid；Tested according to thin-layered chromatography (one B of annex VI of Chinese Pharmacopoeia version in 2010), draw the μ l of need testing solution 20, control medicinal material Solution and each 5 μ l of reference substance solution, put respectively on same silica gel g thin-layer plate, with chloroform-methanol-water (13:7:2) Lower floor's solution is solvent, is deployed, and takes out, dries, spray with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C It is clear；In test sample chromatogram, on position corresponding with control medicinal material chromatogram, show the spot of same color.Ultraviolet lamp is put again Inspected under (365nm)；In test sample chromatogram, on position corresponding with reference substance chromatogram, show the spot of same color；

Check：Meet every regulation (annex IA of Chinese Pharmacopoeia 2010 edition) relevant under pill item.

Assay：

The detection method of content of the albiflorin is：Tested according to Chinese Pharmacopoeia annex high performance liquid chromatography：

The preparation of need testing solution：Zaizao Pill is taken, removes sugar-coat, it is finely ground, 1g is taken, it is accurately weighed, put tool plug taper In bottle, precision adds 50% methanol solution 25mL, close plug, weighed weight, is 250W in power, frequency is at ultrasonic under 35kHz 1h is managed, is let cool, then weighed weight, the weight of less loss is supplied with 50% methanol solution, is shaken up, is filtered, precision measures subsequent filtrate 10mL, water-bath Back stroke is put to 0.5mL, again with methanol dissolving, moves in 10mL measuring bottles, adds methanol to shake up to scale, filter, take Subsequent filtrate, produce；

Determination method：Accurate absorption reference substance solution and each 10ul of need testing solution respectively, injection liquid chromatograph, measure, Produce；

This product is per 1g containing the root of herbaceous peony with Paeoniflorin (C₂₃H₂₈O₁₁) meter, 0.30mg must not be less than；

The detection method of content of the scutelloside is：Tested according to Chinese Pharmacopoeia annex high performance liquid chromatography：

The preparation of need testing solution：Zaizao Pill is taken, removes sugar-coat, it is finely ground, 0.5g is taken, it is accurately weighed, put tool plug cone In shape bottle, precision adds 60% ethanol solution 25mL, close plug, weighed weight, is 250W in power, frequency is ultrasound under 35kHz Processing is ultrasonically treated for 45 minutes, is let cool, then weighed weight, and the weight of less loss is supplied with 60% ethanol solution, is shaken up, and is filtered, is taken Subsequent filtrate, produce：

This product is per 1g containing radix scutellariae with scutelloside (C₂₁H₁₈O₁₁) meter, 2.5mg must not be less than；

The detection method of content of the tanshin polyphenolic acid B is：Tested according to Chinese Pharmacopoeia annex high performance liquid chromatography：

This product is per 1g containing the red sage root with tanshin polyphenolic acid B (C₃₆H₃₀O₁₆) meter, 0.59mg must not be less than.

Inventor has carried out substantial amounts of experiment, is the research of detection method of the present invention below

Experimental example：Detection method research

1 content determination of Baicalin

1.1 instruments and reagent

High performance liquid chromatograph：Wear the peace liquid chromatographs of Ultimate 3000；KQ-500DA type numerical control supersonic extraction apparatuses (Kunshan Ultrasonic Instruments Co., Ltd.)；Zaizao Pill is provided by the auspicious pharmaceutcal corporation, Ltd in Guizhou Dechang；Scutelloside reference substance By the calibrating of Chinese pharmaceutical biological product, institute provides, and lot number is (110715-201117, content is in terms of 91.7%)；Reagent：Methanol (chromatographically pure, analysis are pure), ethanol (analysis is pure), water (the ultra-clean hydrophone of UPW-50N types prepares ultra-pure water), phosphoric acid (analysis is pure).

1.2 assay method

1.2.1 chromatography condition：

(1) chromatographic column：With reference to version pharmacopeia radix scutellariae medicinal materials assay in 2010 select octadecylsilane chemically bonded silica for The pillar of filler is investigated.As a result show to use octadecylsilane chemically bonded silica for filler pillar separating effect compared with It is good.

(2) mobile phase：With reference to version pharmacopeia in 2010, grope by experiment, using the phosphoric acid solution of methanol -0.2% (47:53) For mobile phase, composition scutelloside to be measured and other impurities can obtain preferable separation.

(3) Detection wavelength：Precision weighs appropriate scutelloside reference substance and is dissolved in 60% ethanol solution, takes 60% ethanol as empty White solution, UV scanning wavelength being carried out in 200~400nm wave-length coverages, scutelloside has absorption at 280nm, sees Fig. 1, with This is that Detection wavelength can obtain higher sensitivity and good selection, and with reference to version pharmacopeia radix scutellariae medicinal materials assay in 2010 , thus the Detection wavelength of determination scutelloside is 280nm.

(4) theoretical cam curve：With reference to version pharmacopeia radix scutellariae medicinal materials assay in 2010, theoretical cam curve was based on scutelloside peak Calculate, 2500 should be not less than.

1.2.2 the preparation of need testing solution：

Take this product appropriate, remove sugar-coat, it is finely ground, about 0.5g is taken, it is accurately weighed, put in conical flask with cover, precision adds 60% ethanol solution 25mL, close plug, weighed weight are ultrasonically treated (power 250W, frequency 25kHz) 45 minutes, let cool, then weighed Weight, the weight of less loss is supplied with 60% ethanol solution, is shaken up, filtered, take subsequent filtrate, produce.

1.2.3 the preparation of reference substance solution：

Precision claims scutelloside reference substance appropriate, adds 60% ethanol that the solution that every 1ml contains 50 μ g is made, shakes up, produce.

1.3 method validation

1.3.1 degree of accuracy test solution

Zaizao Pill (5) (average content 4.2251mg/g) the about 0.25g for having determined content is taken, it is accurately weighed, put tool Fill in conical flask, precision adds scutelloside reference substance solution (0.0540113mg/ml) 25ml, by need testing solution preparation method Sample solution is prepared, and by the content of above-mentioned chromatographic condition measure scutelloside, calculates the rate of recovery, the results are shown in Table 1 and degree of accuracy color Spectrogram (Fig. 5), illustrates there is the preferable rate of recovery.

The recovery test data (n=6) of table 1

1.3.2 replica test

Take with a collection of Zaizao Pill (5), prepare 6 parts by need testing solution preparation method, determined by above-mentioned chromatographic condition The content of scutelloside, 2 and repeated chromatogram (Fig. 6) are the results are shown in Table, illustrates there is preferable repeatability.

The replica test of table 2 (n=6)

1.3.3 precision test

Precision measures same scutelloside reference substance solution (53.0393 μ g/ml) 10 μ l, continuous sample introduction 6 times, records chromatogram Figure, the results are shown in Table 3 and precision chromatogram (Fig. 7), illustrates there is preferable precision.

The precision test of table 3 (n=6)

1.3.4 specificity

Take that Zaizao Pill (5) and negative sample prepare sample solution by need testing solution preparation method and negative sample is molten Liquid, by above-mentioned chromatographic condition, reference substance solution, sample solution, negative sample solution liquid is taken to enter liquid chromatograph respectively, sample is molten Liquid chromatography is equipped with corresponding chromatographic peak in reference substance solution chromatogram corresponding positions, and negative noiseless, sees that specificity chromatogram is (specific See Fig. 8-10).

1.3.5 linear relationship is investigated

By above-mentioned chromatographic condition, precision takes scutelloside reference substance (53.0393 μ g/ml) 2 μ l, 5 μ l, 10 μ l, 15 μ l, 25 μ l Liquid chromatograph is implanted sequentially, determines peak area.Using reference substance sample size X as abscissa, peak area Y is ordinate, enters line Property return calculate, drawing curve (Fig. 1 and Fig. 2), show that equation of linear regression is：Y=122.88X-2.14, R=1.Knot Fruit is shown in Table 4 and Figure 11, and the sample size of scutelloside reference substance in the range of the μ g of 0.1061 μ g~1.3260 is with peak area in good Linear relationship.

The scutelloside reference substance linear relationship of table 4

1.3.6 scope

It is measured by sampling by 80% and the 120% of normal measure sampling amount, investigates the precision in height sampling amount measure.Take Zaizao Pill (5) is just sampling each 6 parts, is prepared by need testing solution preparation method, and radix scutellariae is determined by above-mentioned chromatographic condition The content of glycosides, the results are shown in Table 5.

The range finding result of table 5

1.3.7 durability

1.3.7.1 the selection of sample extraction solvent

Zaizao Pill (5) sample is taken, it is finely ground, it is well mixed, takes 6 parts, every part of about 0.5g, it is accurately weighed, tool is put respectively Fill in conical flask, it is each to be separately added into absolute ethyl alcohol, 90% ethanol, 80% ethanol, 70% ethanol, 60% ethanol, 50% ethanol 25ml, ultrasonic (frequency 34KHZ, power 250W) are handled 30 minutes, are taken out, are let cool, supply less loss weight with coordinative solvent, shake It is even, filtration, need testing solution is obtained, take each 10 μ l injections liquid chromatograph of above-mentioned 6 parts of solution respectively, calculate, the results are shown in Table 6, say Bright this product is more complete as Extraction solvent, content extraction using 60% ethanol.

The selection of the Extraction solvent of table 6

1.3.7.2 the selection of sample extraction method

Zaizao Pill (5) sample is taken, it is finely ground, it is well mixed, takes 2 parts, every part of about 0.5g, it is accurately weighed, tool is put respectively Fill in conical flask, precision 60% ethanol 25ml of addition, a copy of it ultrasound (frequency 34KHZ, power 250W) processing 60 minutes, separately A refluxing extraction 60 minutes, take out, let cool, supply less loss weight with 60% ethanol, shake up, filter, take the continuous filters of 10 μ l respectively Liquid injects liquid chromatograph, calculates, and the results are shown in Table 7, ultrasound is with both refluxing extractions content without essential distinction, it is contemplated that ultrasound carries Extract operation is easier, therefore selects ultrasonic extraction.

The selection of the extracting method of table 7

1.3.7.3 sample extraction time effects

Zaizao Pill (5) sample is taken, it is finely ground, it is well mixed, takes 4 parts, every part of about 0.5g, it is accurately weighed, tool is put respectively Fill in conical flask, precision adds 60% ethanol 25ml, and ultrasonic (frequency 34KHZ, power 250W) handles 15,30,45,60 points respectively Clock, take out, let cool, supply less loss weight with 60% ethanol, shake up, filter, take 10 μ l subsequent filtrates to inject liquid chromatograph respectively, Calculate, the results are shown in Table 8 and Figure 12, with ultrasonic 45 minutes for the sample extraction time.

The extraction time of table 8 influences

1.3.7.4 the influence of different brands pillar

Different brands octadecylsilane chemically bonded silica is used to determine Zaizao Pill respectively for the pillar of filler (5) content of scutelloside in, investigates the influence of the chromatographic column of different brands.Chromatogram is shown in durability chromatogram, assay knot Fruit is shown in Table 9 and Figure 13.As a result show chromatographic column of the different brands octadecylsilane chemically bonded silica for filler to assay Without influence.

The measurement result of the different brands chromatographic column of table 9

1.3.7.5 the influence of mobile phase ratio

In the case where not changing mobile phase member condition, change mobile phase ratio, investigate the influence of different proportion mobile phase.As a result Under selected mobile phase ratio, sample separating degree is preferable, and assay the results are shown in Table 10 and Figure 14.

The mobile phase ratio measurement result of table 10

1.3.7.6 the influence of different column temperatures

With same chromatographic column, with same ratio mobile phase, it is measured with different column temperatures, it is 25 DEG C to investigate column temperature respectively, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C can be seen that moving within narrow limits for column temperature does not make significant difference to measurement result, is specifically shown in by the result of table 11 Figure 15.

The influence of 11 different column temperatures of table

3.7.7 sample stability is tested

Zaizao Pill (5) is taken, is prepared by need testing solution preparation method, containing for scutelloside is determined by above-mentioned chromatographic condition Amount, the results are shown in Table 12 and Figure 16.

The sample stability of table 12 is tested

1.4 samples determine

By above-mentioned condition, 10 batches of samples are determined respectively, the results are shown in Table 13 and Figure 17.

The sample measurement result of table 13

Because this product radix scutellariae medicinal materials are used as medicine for crude drug powder, therefore per 1g containing radix scutellariae with scutelloside (C₂₁H₁₈O₁₁) meter, content limit It should be：In prescription radix scutellariae amount × radix scutellariae medicinal materials limit ÷ be made the ÷ 1000g of condensed pill weight × 0.9=32.57g × 9.0% × 1000mg × 0.9=2.6382mg/g.Therefore tentative this product contains radix scutellariae per 1g with scutelloside (C₂₁H₁₈O₁₁) meter, it must not be less than 2.5mg。

2 content of danshinolic acid B determine

2.1 instruments and reagent

High performance liquid chromatograph：Wear the peace liquid chromatographs of Ultimate 3000；Thermostat water bath；Zaizao Pill is by expensive The auspicious pharmaceutcal corporation, Ltd in state Dechang provides；Tanshin polyphenolic acid B reference substance is provided by Chinese pharmaceutical biological product calibrating institute, and lot number is (111562-201212, content is in terms of 95.4%)；Reagent：Methanol (chromatographically pure, analysis are pure), ethanol (analysis is pure), water (UPW- The ultra-clean hydrophone of 50N types prepares ultra-pure water), phosphoric acid (analysis is pure).

2.2 assay method

2.2.1 chromatography condition

(1) chromatographic column：With reference to version pharmacopeia red rooted salvia assay in 2010 select octadecylsilane chemically bonded silica for The pillar of filler is investigated.As a result show to use octadecylsilane chemically bonded silica for filler pillar separating effect compared with It is good.

(2) mobile phase：Grope by experiment, use the phosphoric acid solution of methanol -0.1% (40: 60) as mobile phase, composition to be measured Tanshin polyphenolic acid B and other impurities can obtain preferable separation.

(3) Detection wavelength：Precision weighs appropriate tanshin polyphenolic acid B reference substance and is dissolved in 80% methanol solution, takes 80% methanol conduct Blank solution, UV scanning wavelength being carried out in 190~400nm wave-length coverages, tanshin polyphenolic acid B has absorption at 286nm, sees Fig. 3, Higher sensitivity and good selection can be obtained as Detection wavelength, and with reference to version pharmacopeia red rooted salvia in 2010 containing measurement Determine item, thus the Detection wavelength of determination tanshin polyphenolic acid B is 286nm.

(4) theoretical cam curve：With reference to version pharmacopeia the second enlarged edition red rooted salvia assay in 2010, theoretical cam curve was pressed Tanshin polyphenolic acid B peak calculates, and should be not less than 2000.

2.2.2 the preparation of need testing solution：

Take this product (Zaizao Pill) in right amount, remove sugar-coat, it is finely ground, about 1g is taken, it is accurately weighed, put in conical flask with cover, Precision adds 80% methanol solution 25mL, close plug, weighed weight, is heated to reflux 30 minutes, lets cool, then weighed weight, with 80% Methanol solution supplies the weight of less loss, shakes up, and filtration, takes subsequent filtrate, produces.

2.2.3 the preparation of reference substance solution：

Precision claims tanshin polyphenolic acid B reference substance appropriate, adds 80% methanol that the solution that every 1ml contains 40 μ g is made, shakes up, produce.

2.3 method validation

2.3.1 degree of accuracy test solution

Zaizao Pill (10) (average content 0.8868mg/g) the about 0.5g for having determined content is taken, it is accurately weighed, put tool Fill in conical flask, precision adds tanshin polyphenolic acid B reference substance solution (0.10024632mg/ml) 5ml, then 80% methanol of accurate addition 20ml, from being heated to reflux 30 minutes, sample solution is prepared by need testing solution preparation method, and determine by above-mentioned chromatographic condition The content of tanshin polyphenolic acid B, calculate the rate of recovery, the results are shown in Table 14 and degree of accuracy chromatogram (being specifically shown in Figure 18 and Figure 19), illustrate to have compared with The good rate of recovery.

The recovery test data (n=6) of table 14

2.3.2 replica test

Take with a collection of Zaizao Pill (10), prepare 6 parts by need testing solution preparation method, determined by above-mentioned chromatographic condition The content of tanshin polyphenolic acid B, 15 and repeated chromatogram are the results are shown in Table, illustrates there is preferable repeatability, is specifically shown in Figure 20.

The replica test of table 15 (n=6)

2.3.3 precision test

Precision measures same tanshin polyphenolic acid B reference substance solution (40.098528 μ g/ml) 10 μ l, continuous sample introduction 6 times, records color Spectrogram, as a result table 16 and precision chromatogram, illustrate there is preferable precision, are specifically shown in Figure 21.

The precision test of table 16 (n=6)

2.3.4 specificity

Take that Zaizao Pill (4) and negative sample prepare sample solution by need testing solution preparation method and negative sample is molten Liquid, by above-mentioned chromatographic condition, reference substance solution, sample solution, negative sample solution is taken to enter liquid chromatograph, sample solution respectively Chromatogram is equipped with corresponding chromatographic peak in reference substance solution chromatogram corresponding positions, and negative noiseless, see specificity chromatogram (Figure 22- 24)。

2.3.5 linear relationship is investigated

By above-mentioned chromatographic condition, precision takes tanshin polyphenolic acid B reference substance (40.098528 μ g/ml) 1 μ l, 2 μ l, 5 μ l, 10 μ l, and 15 μ l, 20 μ l are implanted sequentially liquid chromatograph, determine peak area.Using reference substance sample size X as abscissa, peak area Y is ordinate, Linear regression calculating is carried out, drawing curve (Fig. 4), show that equation of linear regression is：Y=918.61X+0.016, R= 0.9999.It the results are shown in Table 17, the sample size of tanshin polyphenolic acid B reference substance in the range of the μ g of 0.040098528 μ g~0.80197056 It is in good linear relationship with peak area, sees Figure 25.

The tanshin polyphenolic acid B reference substance linear relationship of table 17

2.3.6 scope

It is measured by sampling by 80% and the 120% of normal measure sampling amount, investigates the precision in height sampling amount measure.Take Zaizao Pill (10) is just sampling each 6 parts, is prepared by need testing solution preparation method, and red phenol is determined by above-mentioned chromatographic condition Sour B content, the results are shown in Table 18.

The range finding result of table 18

2.3.7 durability

2.3.7.1 the selection of sample extraction solvent

Zaizao Pill (4) sample is taken again, it is finely ground, it is well mixed, takes 7 parts, every part of about 1g, it is accurately weighed, tool is put respectively Fill in conical flask, it is accurate respectively to add ethanol, methanol, 90% methanol, 80% methanol, 70% methanol, 60% methanol, 50% methanol Each 25ml, it is heated to reflux 30 minutes, takes out, let cool, supply less loss weight with coordinative solvent, shake up, filters, it is molten to obtain test sample Liquid, each 10 μ l injections liquid chromatograph of above-mentioned 2 parts of solution is taken respectively, is calculated, be the results are shown in Table 19, Figure 26 and Figure 27, illustrate this product It is more complete as Extraction solvent, content extraction using 80% methanol.

The selection of the Extraction solvent of table 19

2.3.7.2 the selection of sample extraction method

Zaizao Pill (4) sample is taken, it is finely ground, it is well mixed, takes 2 parts, every part of about 1g, it is accurately weighed, tool plug is put respectively In conical flask, precision adds 80% methanol 25ml, and a copy of it ultrasound (frequency 34KHZ, power 250W) is handled 30 minutes, another Part refluxing extraction 30 minutes, takes out, lets cool, supply less loss weight with 80% methanol, shake up, and filters, takes 10 μ l subsequent filtrates respectively Liquid chromatograph is injected, calculates, the results are shown in Table 20, illustrates that this product uses refluxing extraction, content is extracted more complete.

The selection of the extracting method of table 20

2.3.7.3 sample extraction time effects

Zaizao Pill (4) sample is taken, it is finely ground, it is well mixed, takes 4 parts, every part of about 1g, it is accurately weighed, tool plug is put respectively In conical flask, precision add 80% methanol 25ml, be heated to reflux respectively processing 15,30,60,120 minutes, take out, let cool, use 80% methanol supplies less loss weight, shakes up, and filtration, takes 10 μ l subsequent filtrates to inject liquid chromatograph respectively, calculates, the results are shown in Table 21 And Figure 28, the sample extraction time is used as in 30 minutes using backflow.

The extraction time of table 21 influences

2.3.7.4 the influence of different brands pillar

Different brands octadecylsilane chemically bonded silica is used to determine Zaizao Pill respectively for the pillar of filler (4) content of tanshin polyphenolic acid B in, investigates the influence of the chromatographic column of different brands.Chromatogram is shown in durability chromatogram, assay knot Fruit is shown in Table 22 and Figure 29.As a result show chromatographic column of the different brands octadecylsilane chemically bonded silica for filler to assay Without influence.

The measurement result of the different brands chromatographic column of table 22

2.3.7.5 the influence of mobile phase ratio

In the case where not changing mobile phase member condition, change mobile phase ratio, investigate the influence of different proportion mobile phase.As a result Under selected mobile phase ratio, sample separating degree is preferable, and assay the results are shown in Table 23 and Figure 30.

The mobile phase ratio measurement result of table 23

2.3.7.6 the influence of different column temperatures

With same chromatographic column, with same ratio mobile phase, it is measured with different column temperatures, it is 20 DEG C to investigate column temperature respectively, 30 DEG C, 40 DEG C, chromatogram is shown in durability chromatogram (Figure 31), can be seen that by the result of table 24, and moving within narrow limits for column temperature is tied to measure Fruit does not make significant difference.

The different column temperature measurement results of table 24

2.3.7.7 sample stability is tested

Zaizao Pill (10) is taken, is prepared by need testing solution preparation method, scutelloside is determined by above-mentioned chromatographic condition Content, it the results are shown in Table 25.

The sample stability of table 25 is tested

2.4 samples determine

By above-mentioned condition, 10 batches of samples are determined respectively, the results are shown in Table 26 and Figure 32.

The sample measurement result of table 26

Because this product red rooted salvia is used as medicine for crude drug powder, therefore per 1g containing the red sage root with tanshin polyphenolic acid B (C₃₆H₃₀O₁₆) meter, content limit Degree should be：The ÷ 1000g of condensed pill weight × 0.9=21.71g × 3.0% are made in red sage root amount × red rooted salvia limit ÷ in prescription × 1000mg × 0.9=0.5862mg/g.Therefore tentative this product contains the red sage root per 1g with tanshin polyphenolic acid B (C₃₆H₃₀O₁₆) meter, it must not be less than 0.59mg。

The beneficial effects of the present invention are：The invention provides a kind of detection method of Zaizao Pill, including with effective Composition albiflorin, scutelloside and/or tanshin polyphenolic acid B are index, the content of measure albiflorin, scutelloside and/or tanshin polyphenolic acid B；It is described Detection method is accurate, high sensitivity, reproducible, and testing result is stable, can effectively control the quality of Zaizao Pill, both more have Beneficial to the monitoring of manufacturer and supervisory and management department to product quality, or the treatment of medical department and patient provide more Good guarantee.

Brief description of the drawings

When Fig. 1 is scutelloside detection, uv scan figure；

Fig. 2 is scutelloside linear graph；

When Fig. 3 is tanshin polyphenolic acid B detection, uv scan figure；

Fig. 4 is the system suitability test figure of Gastrodin reference substance；

Fig. 5 is scutellaria glycosides reference substance degree of accuracy chromatogram；

Fig. 6 is Zaizao Pill scutellaria glycosides accuracy of measurement chromatogram；

When Fig. 7 is scutellaria glycosides measure, precision chromatogram；

When Fig. 8 is scutellaria glycosides measure, negative sample specificity chromatogram；

When Fig. 9 is scutellaria glycosides measure, reference substance specificity chromatogram；

When Figure 10 is scutellaria glycosides in measure Zaizao Pill, specificity chromatogram；

When Figure 11 is scutellaria glycosides measure, linear relationship investigates figure；

Figure 12 is the timing of scutellaria glycosides side, and different extracting modes and time investigate result figure；

Figure 13 be scutellaria glycosides side timing, the influence figure of different brands pillar；

Figure 14 be scutellaria glycosides side timing, the influence figure of mobile phase ratio；

Figure 15 be scutellaria glycosides side timing, the influence figure of different column temperatures；

When Figure 16 is scutellaria glycosides assay, stability test result figure；

Figure 17 is scutellaria glycosides assay result figure；

When Figure 18 is content of danshinolic acid B measure, tanshin polyphenolic acid B reference substance degree of accuracy chromatogram；

When Figure 19 is content of danshinolic acid B measure, Zaizao Pill sample degree of accuracy chromatogram；

When Figure 20 is content of danshinolic acid B measure, replica test chromatogram；

When Figure 21 is content of danshinolic acid B measure, precision chromatogram；

When Figure 22 is content of danshinolic acid B measure, reference substance tanshin polyphenolic acid B specificity chromatogram；

When Figure 23 is content of danshinolic acid B measure, negative sample specificity chromatogram；

When Figure 24 is content of danshinolic acid B measure, Zaizao Pill sample specificity chromatogram；

When Figure 25 is content of danshinolic acid B measure, linear relationship spectrogram；

When Figure 26 is content of danshinolic acid B measure, alcohol reflux extraction result figure；

When Figure 27 is content of danshinolic acid B measure, methanol eddy extraction result figure；

When Figure 28 is content of danshinolic acid B measure, refluxing extraction 30min result figures；

When Figure 29 is content of danshinolic acid B measure, different pillar durability chromatograms；

When Figure 30 is content of danshinolic acid B measure, mobile phase ratio influences result figure；

When Figure 31 is content of danshinolic acid B measure, different column temperature durability chromatograms；

Figure 32 is content of danshinolic acid B result figure in Zaizao Pill sample.

With reference to embodiment, the present invention is further illustrated

Embodiment

Embodiment 1：

Zaizao Pill prescription：Prepared RADIX ANGELICAE SINENSIS with yellow rice wine 65.14g, prepared RHIZOMA CYPERI with vinegar 65.14g, root of herbaceous peony 43.43g, prepared rhizome of rehmannia 21.71g, donkey-hide gelatin 10.86g, Poria cocos 65.14g, Radix Codonopsis 21.71g, Radix Astragali 21.71g, Chinese yam 32.57g, bighead atractylodes rhizome 16.28g, wine fruit of glossy privet 43.43g, Tortoise plastron (vinegar is processed) 32.57g, Fructus Corni 21.71g, teasel root 21.71g, stir-baked CORTEX EUCOMMIAE with salt solution 21.71g, saline cistanche 10.86g, raspberry 16.28g, cornu cerve degelatinatum 5.43g, Ligusticum wallichii 43.43g, red sage root 21.71g, root of bidentate achyranthes 16.28g, motherwort 21.71g, corydalis tuber 16.28g, pseudo-ginseng (short) 5.43g, folium artemisiae argyi (vinegar is processed) 43.43g, fennel seeds 21.71g, Jehol Ligusticum Rhizome 21.71g, cuttlebone 32.57g, Garden burnet (wine is processed) 32.57g, intelligence development 10.86g, rhizoma alismatis 21.71g, lotus leaf (vinegar is processed) 16.28g, bark of ash 21.71g, the root bark of Chinese wolf-berry 21.71g, radix cynanchi atrati 43.43g, bark of tree of heaven 32.57g, amber 5.43g, stir-baked RADIX SCUTELLARIAE with yellow rice wine 32.57g, spina date seed 10.86g, RADIX POLYGALAE PREPARATA 16.28g, dried orange peel 32.57g and radix glycyrrhizae 21.71g.

Zaizao Pill preparation method：The taste medicine of the above 42, the Radix Astragali, stir-baked CORTEX EUCOMMIAE with salt solution, prepared rhizome of rehmannia, teasel root, bark of ash, saline cistanche, ox Knee, the root bark of Chinese wolf-berry add water to cook 2 times, 2 hours every time, collecting decoction, filter, filtrate concentration；It is another take donkey-hide gelatin add suitable quantity of water molten with Above-mentioned concentrate merges, and is concentrated into the clear cream that relative density is 1.10 (80 DEG C).The taste of remaining Radix Angelicae Sinensis etc. 33 is ground into fine powder, Sieving, mix；With the general ball of above-mentioned clear cream, condensed pill 1000g is made, 60-80 DEG C of drying, sugar coating, polishing, produces Zaizao Ball.

Assay：

Claims

A kind of 1. detection method of Zaizao Pill, it is characterised in that：The medicine is by prepared RADIX ANGELICAE SINENSIS with yellow rice wine 60-70 parts, prepared RHIZOMA CYPERI with vinegar 60- 70 parts, root of herbaceous peony 42-45 parts, prepared rhizome of rehmannia 20-23 parts, donkey-hide gelatin 9-12 parts, Poria cocos 60-70 parts, Radix Codonopsis 20-23 parts, Radix Astragali 20-23 Part, Chinese yam 30-35 parts, bighead atractylodes rhizome 15-17 parts, wine fruit of glossy privet 42-45 parts, vinegar process tortoise plastron 30-35 parts, Fructus Corni 20-23 parts, teasel root 20-23 parts, stir-baked CORTEX EUCOMMIAE with salt solution 20-23 parts, saline cistanche 9-12 parts, raspberry 15-17 parts, cornu cerve degelatinatum 3-7 parts, Ligusticum wallichii 42-45 parts, the red sage root 20-23 parts, root of bidentate achyranthes 15-17 parts, motherwort 20-23 parts, corydalis tuber 15-17 parts, short pseudo-ginseng 3-7 parts, vinegar process folium artemisiae argyi 42-45 Part, fennel seeds 20-23 parts, Jehol Ligusticum Rhizome 20-23 parts, cuttlebone 30-35 parts, wine process garden burnet 30-35 parts, intelligence development 9-12 parts, rhizoma alismatis 20- 23 parts, vinegar process lotus leaf 15-17 parts, bark of ash 20-23 parts, root bark of Chinese wolf-berry 20-23 parts, radix cynanchi atrati 42-45 parts, bark of tree of heaven 30-35 parts, amber 3- 7 parts, stir-baked RADIX SCUTELLARIAE with yellow rice wine 30-35 parts, spina date seed 9-12 parts, RADIX POLYGALAE PREPARATA 15-17 parts, dried orange peel 30-35 parts and radix glycyrrhizae 20-23 parts are under The method of stating is prepared：The taste medicine of the above 42, the Radix Astragali, stir-baked CORTEX EUCOMMIAE with salt solution, prepared rhizome of rehmannia, teasel root, bark of ash, saline cistanche, the root of bidentate achyranthes, the root bark of Chinese wolf-berry Add water to cook 2 times, 2 hours every time, collecting decoction, filter, filtrate concentration；Separately donkey-hide gelatin is taken to add suitable quantity of water molten and above-mentioned concentrate Merge, be concentrated into the clear cream that relative density at 78-82 DEG C is 1.10；The taste of remaining Radix Angelicae Sinensis etc. 33 is ground into fine powder, sieves, and mixes It is even；With the general ball of above-mentioned clear cream, condensed pill 1000g is made, 60-80 DEG C of drying, sugar coating, polishing, pill is made；The detection side Method includes content assaying method；The content assaying method includes the assay to albiflorin, scutelloside and/or tanshin polyphenolic acid B.
2. the detection method of Zaizao Pill as claimed in claim 1, it is characterised in that：The detection method of content of the albiflorin For：Tested according to Chinese Pharmacopoeia annex high performance liquid chromatography：

Chromatographic condition and system suitability：Using cyano group bonded silica gel as filler；With methanol-water（11：86-92）For stream Dynamic phase;Detection wavelength is 230nm；Theoretical cam curve is calculated by Paeoniflorin peak, should be not less than 4000；

The preparation of reference substance solution：Paeoniflorin reference substance 8-12mg is taken, it is accurately weighed, put in 20mL measuring bottles, add methanol dissolving simultaneously Scale is diluted to, is shaken up, precision measures 0.8-1.2mL, puts in 20mL measuring bottles, adds methanol to shake up to scale, produce every 1mL and contain Chinese herbaceous peony 23-27ug reference substance solution；

The preparation of need testing solution：Take this product appropriate, remove sugar-coat, it is finely ground, 0.8-1.2g is taken, it is accurately weighed, put tool plug taper In bottle, precision adds 45-55% methanol solutions 23-27mL, close plug, weighed weight, is ultrasonically treated, lets cool, then weighed weight, uses 45-55% methanol solutions supply the weight of less loss, shake up, and filtration, precision measures subsequent filtrate 10mL, puts water-bath Back stroke to 0.4- 0.6mL, again with methanol dissolving, is moved in 10mL measuring bottles, adds methanol to shake up to scale, filter, take subsequent filtrate, produce；

Determination method：It is accurate respectively to draw reference substance solution and each 10ul of need testing solution, liquid chromatograph is injected, measure, is produced.
3. the detection method of Zaizao Pill as claimed in claim 2, it is characterised in that：The detection method of content of the albiflorin For：Tested according to Chinese Pharmacopoeia annex high performance liquid chromatography：

Chromatographic condition and system suitability：Using cyano group bonded silica gel as filler；With methanol-water（11：89）For mobile phase; Detection wavelength is 230nm；Theoretical cam curve is calculated by Paeoniflorin peak, should be not less than 4000；

The preparation of reference substance solution：Paeoniflorin reference substance 10mg is taken, it is accurately weighed, put in 20mL measuring bottles, add methanol to dissolve and dilute Release to scale, shake up, precision measures 1mL, puts in 20mL measuring bottles, adds methanol to shake up to scale, produce every 1mL 25ug containing Chinese herbaceous peony Reference substance solution；

The preparation of need testing solution：Zaizao Pill is taken, removes sugar-coat, it is finely ground, 1g is taken, it is accurately weighed, put in conical flask with cover, Precision adds 50% methanol solution 25mL, close plug, weighed weight, is ultrasonically treated, lets cool, then weighed weight, with 50% methanol solution The weight of less loss is supplied, is shaken up, is filtered, precision measures subsequent filtrate 10mL, puts water-bath Back stroke to 0.5mL, again with methanol dissolving, moves Into 10mL measuring bottles, methanol is added to shake up to scale, filter, take subsequent filtrate, produce；

Determination method：It is accurate respectively to draw reference substance solution and each 10ul of need testing solution, liquid chromatograph is injected, measure, is produced.
4. the detection method of Zaizao Pill as claimed in claim 2 or claim 3, it is characterised in that：The supersound process is；In work( Rate is 245-255W, and frequency is to be ultrasonically treated 0.8-1.2h under 33-37kHz.
5. the detection method of Zaizao Pill as claimed in claim 1, it is characterised in that：The content detection side of the scutelloside Method is：Tested according to Chinese Pharmacopoeia annex high performance liquid chromatography：

Chromatographic condition and system suitability test：Using octadecylsilane chemically bonded silica as filler；It is molten with the phosphoric acid of methanol -0.2% Liquid（47:50-56）For mobile phase：Detection wavelength is 275-285nm；Theoretical cam curve is calculated by scutelloside peak, should be not less than 2500；

The preparation of reference substance solution：Precision claims scutelloside reference substance appropriate, adds 55-65% ethanol to be made every 1ml μ containing 48-52 g's Solution, shake up, produce；

The preparation of need testing solution：Zaizao Pill is taken, removes sugar-coat, it is finely ground, 0.5g is taken, it is accurately weighed, put conical flask with cover In, precision adds 55-65% ethanol solutions 23-27mL, close plug, weighed weight, is ultrasonically treated, lets cool, then weighed weight, uses 55- 65% ethanol solution supplies the weight of less loss, shakes up, and filtration, takes subsequent filtrate, produces；

Determination method：It is accurate respectively to draw reference substance solution and each 10ul of need testing solution, liquid chromatograph is injected, measure, is produced.
6. the detection method of Zaizao Pill as claimed in claim 5, it is characterised in that：The content detection side of the scutelloside Method is：Tested according to Chinese Pharmacopoeia annex high performance liquid chromatography：

Chromatographic condition and system suitability test：Using octadecylsilane chemically bonded silica as filler；It is molten with the phosphoric acid of methanol -0.2% Liquid（47:53）For mobile phase：Detection wavelength is 280m；Theoretical cam curve is calculated by scutelloside peak, should be not less than 2500；

The preparation of reference substance solution：Precision claims scutelloside reference substance appropriate, adds 60% ethanol that the solution that every 1ml contains 50 μ g is made, shakes It is even, produce；

The preparation of need testing solution：Zaizao Pill is taken, removes sugar-coat, it is finely ground, 0.5g is taken, it is accurately weighed, put conical flask with cover In, precision adds 60% ethanol solution 25mL, close plug, weighed weight, is ultrasonically treated, lets cool, then weighed weight, molten with 60% ethanol Liquid supplies the weight of less loss, shakes up, and filtration, takes subsequent filtrate, produces：

Determination method：It is accurate respectively to draw reference substance solution and each 10ul of need testing solution, liquid chromatograph is injected, measure, is produced.
7. the detection method of the Zaizao Pill as described in claim 5 or 6, it is characterised in that：The supersound process is；In work( Rate is 245-255W, and frequency is that 40-50 minutes are ultrasonically treated under 30-40kHz.
8. the detection method of Zaizao Pill as claimed in claim 1, it is characterised in that：The content detection of the tanshin polyphenolic acid B Method is：Tested according to Chinese Pharmacopoeia annex high performance liquid chromatography：

Chromatographic condition and system suitability test：Using octadecylsilane chemically bonded silica as filler；It is molten with the phosphoric acid of methanol -0.1% Liquid（40：55-65）For mobile phase；Detection wavelength is 285-287nm；Theoretical cam curve is calculated by tanshin polyphenolic acid B peak, should be not less than 2000；

The preparation of reference substance solution：Precision claims tanshin polyphenolic acid B reference substance, adds 75-85% methanol that the molten of every 1ml μ containing 38-42 g is made Liquid, shake up, produce；

The preparation of need testing solution：Zaizao Pill is taken, removes sugar-coat, it is finely ground, 1g is taken, it is accurately weighed, put in conical flask with cover, Precision adds 75-85% methanol solutions 20-30mL, close plug, weighed weight, is heated to reflux 25-35 minutes, lets cool, then weighed heavy Amount, the weight of less loss is supplied with 78-82% methanol solutions, is shaken up, and is filtered, is taken subsequent filtrate, produce；

Determination method：It is accurate respectively to draw reference substance solution and each 10ul of need testing solution, liquid chromatograph is injected, measure, is produced.
9. the detection method of Zaizao Pill as claimed in claim 1, it is characterised in that：The content detection of the tanshin polyphenolic acid B Method is：Tested according to Chinese Pharmacopoeia annex high performance liquid chromatography：

Chromatographic condition and system suitability test：Using octadecylsilane chemically bonded silica as filler；It is molten with the phosphoric acid of methanol -0.1% Liquid（40：60）For mobile phase；Detection wavelength is 286nm；Theoretical cam curve is calculated by tanshin polyphenolic acid B peak, should be not less than 2000；

The preparation of reference substance solution：Precision claims tanshin polyphenolic acid B reference substance, adds 80% methanol that the solution that every 1ml contains 40 μ g is made, shakes up, Produce；

The preparation of need testing solution：Zaizao Pill is taken, removes sugar-coat, it is finely ground, 1g is taken, it is accurately weighed, put in conical flask with cover, Precision adds 80% methanol solution 25mL, close plug, weighed weight, is heated to reflux 30 minutes, lets cool, then weighed weight, with 80% first Alcoholic solution supplies the weight of less loss, shakes up, and filtration, takes subsequent filtrate, produces；

Determination method：It is accurate respectively to draw reference substance solution and each 10ul of need testing solution, liquid chromatograph is injected, measure, is produced.
10. the detection method of Zaizao Pill as claimed in claim 1, it is characterised in that：The medicine so detects：

Character：This product is sugar-coat condensed pill, removes sugar-coat and shows brown color to sepia；Mildly bitter flavor, slightly fiber crops；

Differentiate：（1）This product is taken, puts micro- Microscopic observation：Irregular branched agglomerate is colourless, meets chloraldurate liquid and dissolves；Mycelia Colourless or light brown, 38 μm of diameter（Poria cocos）；Fiber is faint yellow, fusiformis, wall thickness, and hole ditch is thin（Radix scutellariae）；T-shaped nonglandular hair is curved Song, 24 cells of handle, often comes off（Folium artemisiae argyi）；

（2）This product 15g is taken, it is finely ground, add petroleum ether（60~90℃）50ml, it is heated to reflux 40 minutes, filters, filtrate volatilizes, residue Ethyl acetate 1ml is added to make dissolving, as need testing solution；α-cyperolone reference substance separately is taken, adds ethyl acetate that every 1ml is made and contains 1mg solution, as reference substance solution；According to thin-layered chromatography（One B of annex VI of Chinese Pharmacopoeia version in 2010）Experiment, draw μ l of need testing solution 10, the μ l of reference substance solution 2, put respectively on same silica gel g thin-layer plate, making into strips, with hexamethylene-acetic acid Ethyl ester（9:1）For solvent, deploy, take out, dry, spray with dinitrophenylhydrazine test solution, place 1-5min；In test sample chromatogram, On position corresponding with reference substance chromatogram, show the orange red spot of identical；

（3）This product 15g is taken, it is finely ground, add methanol 50ml, be heated to reflux 45 minutes, let cool, filter, filtrate is evaporated, and residue adds water 30ml makes dissolving, with ether shaking extraction 2 times, each 20ml, discards ether liquid, shakes extraction 3 times with water saturated n-butanol, often Secondary 25ml, merge n-butanol liquid, then the washing 3 times with n-butanol saturation, each 20ml, take n-butanol liquid, be evaporated, residue adds first Alcohol 1ml makes dissolving, adds neutral alumina 2g, is mixed thoroughly in water-bath, dries, and loads neutral alumina column（100 ~ 200 mesh, 2g are interior Footpath 10mm, dry column-packing）On, use acetate-methanol（1:1）40ml is eluted, and is collected eluent, is evaporated, residue adds ethanol 1ml Make dissolving, as need testing solution；Paeoniflorin reference substance separately is taken, adds ethanol that solution of every 1ml containing 1mg is made, as reference substance Solution；According to thin-layered chromatography（One B of annex VI of Chinese Pharmacopoeia version in 2010）Experiment, each 10 μ l of above two solution are drawn, point Other point is on same silica gel g thin-layer plate, with chloroform-acetate-methanol-formic acid（40:5:10:0.2）For solvent, exhibition Open, take out, dry, spray with 5% vanillin-sulfuric acid solution, it is clear to be heated to spot development at 105 DEG C；

In test sample chromatogram, on position corresponding with reference substance chromatogram, show the spot of same color；

（4）This product 15g is taken, finely ground, add diethyl ether 50ml, is heated to reflux 30 minutes, filtration, discards ether liquid；The dregs of a decoction fling to ether, add Methanol 50ml, it is heated to reflux 45 minutes, filters, filtrate is evaporated, and residue adds water 25ml to make dissolving, and filtration, filtrate is adjusted with hydrochloric acid PH value, with ethyl acetate shaking extraction 2 times, each 20ml, combined ethyl acetate liquid, is evaporated, it is molten that residue adds methanol 1ml to make to 2 Solution, as need testing solution；Scutelloside reference substance separately is taken, adds methanol that solution of every 1ml containing 1mg is made, as reference substance solution； According to thin-layered chromatography（One B of annex VI of Chinese Pharmacopoeia version in 2010）Experiment, each 10 μ l of above two solution are drawn, respectively point In same on the silica gel g thin-layer plate of 4% sodium acetate, to make into strips, with ethyl acetate-butanone-formic acid-water（5:3:1:1）For exhibition Agent is opened, puts presaturation 30 minutes in expansion cylinder, is deployed, is taken out, is dried, spray with 5% ferric trichloride ethanol solution；

In test sample chromatogram, on position corresponding with reference substance chromatogram, show the spot of same color；

（5）Aurantiamarin reference substance is taken, adds methanol that saturated solution is made, as reference substance solution；According to thin-layered chromatography（Chinese Pharmacopoeia One B of annex VI of version in 2010）Experiment, draw [discriminating]（4）Need testing solution and each 10 μ of above-mentioned reference substance solution under L, put respectively in same on the silica gel g thin-layer plate of 1% sodium hydroxide, to make into strips, with chloroform-methanol-water（32:17: 5）Lower floor's solution be solvent, put presaturation 30 minutes in expansion cylinder, deploy, take out, dry；Spray with alchlor test solution, Put ultraviolet lamp（365nm）Under inspect；In test sample chromatogram, on position corresponding with reference substance chromatogram, show same color Fluorescence spot；

（6）25 g of this product are taken, finely ground, add diethyl ether 80ml, is ultrasonically treated 30 minutes, and filtration, filtrate volatilizes, and residue adds acetic acid second Ester 2ml makes dissolving, as need testing solution；

Red sage root control medicinal material separately is taken, is made in the same way of control medicinal material solution；Tanshinone IIA reference substance is taken again, adds ethyl acetate to be made Per solution of the 1ml containing 1mg, as reference substance solution；According to thin-layered chromatography（One B of annex VI of Chinese Pharmacopoeia version in 2010）Examination Test, draw the μ l of need testing solution 20, control medicinal material solution and each 5 μ l of reference substance solution, put respectively in same silica gel g thin-layer plate On, with benzene-ethyl acetate（19:1）For solvent, deploy, take out, dry；

In test sample chromatogram, on position corresponding with control medicinal material and reference substance chromatogram, show the spot of same color；

（7）This product 25g is taken, it is finely ground, add water saturated n-butanol 80ml, be ultrasonically treated 30 minutes, filtration, filtrate ammonification test solution shakes Extraction 2 times, each 25ml is shaken, discards ammoniacal liquor, n-butanol liquid with the water washing 2 times of n-butanol saturation, each 25ml, discards water again Liquid, n-butanol liquid are evaporated, and residue adds methanol 2ml to make dissolving, as need testing solution；Radix Astragali control medicinal material separately is taken, is made in the same way of Control medicinal material solution；Astragaloside IV reference substance is taken again, adds methanol that solution of every 1ml containing 1mg is made, as reference substance solution；According to Thin-layered chromatography（One B of annex VI of Chinese Pharmacopoeia version in 2010）Experiment, draw the μ l of need testing solution 20, control medicinal material solution With each 5 μ l of reference substance solution, put respectively on same silica gel g thin-layer plate, with chloroform-methanol-water（13:7:2）Lower floor Solution is solvent, is deployed, and takes out, dries, spray with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C；For In test product chromatogram, on position corresponding with control medicinal material chromatogram, show the spot of same color；

Ultraviolet lamp is put again（365nm）Under inspect；In test sample chromatogram, on position corresponding with reference substance chromatogram, show identical The spot of color；

Check：Meet every regulation relevant under pill item（Annex I A of Chinese Pharmacopoeia 2010 edition）；

Assay：

The detection method of content of the albiflorin is：Tested according to Chinese Pharmacopoeia annex high performance liquid chromatography：

Chromatographic condition and system suitability：Using cyano group bonded silica gel as filler；With methanol-water（11：89）For mobile phase; Detection wavelength is 230nm；Theoretical cam curve is calculated by Paeoniflorin peak, should be not less than 4000；

The preparation of reference substance solution：Paeoniflorin reference substance 10mg is taken, it is accurately weighed, put in 20mL measuring bottles, add methanol to dissolve and dilute Release to scale, shake up, precision measures 1mL, puts in 20mL measuring bottles, adds methanol to shake up to scale, produce every 1mL 25ug containing Chinese herbaceous peony Reference substance solution；

The preparation of need testing solution：Zaizao Pill is taken, removes sugar-coat, it is finely ground, 1g is taken, it is accurately weighed, put in conical flask with cover, Precision adds 50% methanol solution 25mL, close plug, weighed weight, is 250W in power, frequency is to be ultrasonically treated 1h under 35kHz, is put It is cold, then weighed weight, the weight of less loss is supplied with 50% methanol solution, is shaken up, is filtered, precision measures subsequent filtrate 10mL, puts water-bath Back stroke again with methanol dissolving, moves in 10mL measuring bottles to 0.5mL, adds methanol to shake up to scale, filter, take subsequent filtrate, produce；

Determination method：It is accurate respectively to draw reference substance solution and each 10ul of need testing solution, liquid chromatograph is injected, measure, is produced；

This product is per 1g containing the root of herbaceous peony with Paeoniflorin（C₂₃H₂₈O₁₁）Meter, must not be less than 0.30mg；

The detection method of content of the scutelloside is：Tested according to Chinese Pharmacopoeia annex high performance liquid chromatography：

Chromatographic condition and system suitability test：Using octadecylsilane chemically bonded silica as filler；It is molten with the phosphoric acid of methanol -0.2% Liquid（47:53）For mobile phase：Detection wavelength is 280m；Theoretical cam curve is calculated by scutelloside peak, should be not less than 2500；

The preparation of reference substance solution：Precision claims scutelloside reference substance appropriate, adds 60% ethanol that the solution that every 1ml contains 50 μ g is made, shakes It is even, produce；

The preparation of need testing solution：Zaizao Pill is taken, removes sugar-coat, it is finely ground, 0.5g is taken, it is accurately weighed, put conical flask with cover In, precision adds 60% ethanol solution 25mL, close plug, weighed weight, is 250W in power, frequency is to be ultrasonically treated 45 under 35kHz Minute is ultrasonically treated, and lets cool, then weighed weight, and the weight of less loss is supplied with 60% ethanol solution, is shaken up, and is filtered, is taken subsequent filtrate, Produce：

Determination method：It is accurate respectively to draw reference substance solution and each 10ul of need testing solution, liquid chromatograph is injected, measure, is produced；

This product is per 1g containing radix scutellariae with scutelloside（C₂₁H₁₈O₁₁）Meter, must not be less than 2.5mg；

The detection method of content of the tanshin polyphenolic acid B is：Tested according to Chinese Pharmacopoeia annex high performance liquid chromatography：

Chromatographic condition and system suitability test：Using octadecylsilane chemically bonded silica as filler；It is molten with the phosphoric acid of methanol -0.1% Liquid（40：60）For mobile phase；Detection wavelength is 286nm；Theoretical cam curve is calculated by tanshin polyphenolic acid B peak, should be not less than 2000；

The preparation of reference substance solution：Precision claims tanshin polyphenolic acid B reference substance, adds 80% methanol that the solution that every 1ml contains 40 μ g is made, shakes up, Produce；

The preparation of need testing solution：Zaizao Pill is taken, removes sugar-coat, it is finely ground, 1g is taken, it is accurately weighed, put in conical flask with cover, Precision adds 80% methanol solution 25mL, close plug, weighed weight, is heated to reflux 30 minutes, lets cool, then weighed weight, with 80% first Alcoholic solution supplies the weight of less loss, shakes up, and filtration, takes subsequent filtrate, produces；

Determination method：It is accurate respectively to draw reference substance solution and each 10ul of need testing solution, liquid chromatograph is injected, measure, is produced；

This product is per 1g containing the red sage root with tanshin polyphenolic acid B（C₃₆H₃₀O₁₆）Meter, must not be less than 0.59mg.