CN105372338A

CN105372338A - Compound yinlingtong capsule quality detection method

Info

Publication number: CN105372338A
Application number: CN201410390074.0A
Authority: CN
Inventors: 冯鹏; 冯敏; 沈建; 钱一帆; 陈素玲; 朱韵韵
Original assignee: NANJING ZHONGKE PHARMACEUTICAL CO Ltd
Current assignee: NANJING ZHONGKE PHARMACEUTICAL CO Ltd
Priority date: 2014-08-08
Filing date: 2014-08-08
Publication date: 2016-03-02

Abstract

The present invention discloses a compound yinlingtong capsule quality detection method, which provides thin-layer identification on ganoderma lucidum, radix scrophulariae and acorus tatarinowii, wherein a high-performance liquid chromatography method is used to carry out content determination on ginkgolide A, ginkgolide B, ginkgolide C and bilobalide. According to the present invention, when the quality control method is used to carry out separation detection, advantages of interference factor resisting, high detection efficiency, good stability, rapid analysis and short detection period can be provided; and the method is the reliable compound yinlingtong capsule control method, and establishes the accurate and feasible control standard for the compound yinlingtong capsule quality detection.

Description

The quality determining method of compound silver Lingtong capsule

Technical field

The invention belongs to technical field of traditional Chinese medicines, be specifically related to the quality determining method of a kind of compound silver Lingtong capsule.

Background technology

Compound silver Lingtong capsule comprises glossy ganoderma, ginkgo leaf, Radix Achyranthis Bidentatae, radix scrophulariae, grass-leaved sweetflag and rhizoma Gastrodiae six kinds of drug components, be rich in GINKGO BILOBA EXTRACT, lactone, ganoderan and triterpene compound, fundamentally can nurse one's health brain, eliminate forgetful, headache, the brain symptom such as dizzy, prevention and alleviate the brain such as apoplexy, senile dementia chronic illness energetically, be the crowd that drinks of the crowd of requiring mental skill and Chang protect brain, product for invigorating function of brain.

Inventor analyzes existing quality standard, finds to there is following problem: 1) method is single, not easily accurately; 2) content assaying method adopts thin layer chromatography scanning, and reappearance is poor, and the impact by point sample environment is comparatively large, and by the impact of extraneous humiture during its thin-layer developing, disturbing factor is more; And the more difficult effective separation of material that structure is similar, round of visits is longer, and the quality control that the present invention is directed to compound silver Lingtong capsule is carried out perfect further.

Summary of the invention

The object of this invention is to provide a kind of method that energy accurately, fast detects compound silver Lingtong capsule quality.

The preparation method of compound silver Lingtong capsule can be as follows:

Formula: glossy ganoderma 300-400 weight portion ginkgo leaf 300-400 weight portion Radix Achyranthis Bidentatae 200-290 weight portion radix scrophulariae 200-290 weight portion grass-leaved sweetflag 150-190 weight portion rhizoma Gastrodiae 100-140 weight portion

Preparation process:

A, grass-leaved sweetflag is ground into particle;

B, by grass-leaved sweetflag particle adopt CO ₂supercritical extract, extracting pressure is 22-30Mpa, and extraction kettle temperature is 45-60 DEG C, and extraction time is 0.5-1.5 hour, obtains volatile oil and grass-leaved sweetflag extract remainder;

C, get the isopyknic ethanol of volatile oil and dissolve, obtain volatile oil ethanolic solution; Separately get the beta-schardinger dextrin-of volatile oil weight 4-8 times amount, grind well with the water of beta-schardinger dextrin-3-5 times amount, pour in colloid mill, slowly drip above-mentioned volatile oil ethanolic solution continuously, mill inclusion 20-40 minute, inclines and, 0-4 DEG C of refrigeration leaves standstill more than 24 hours, filter, in 40-80 DEG C of vacuum drying, obtain volatile oil clathrate compound;

It is the ethanol of 70-90% that d, glossy ganoderma, ginkgo leaf, Radix Achyranthis Bidentatae, radix scrophulariae and rhizoma Gastrodiae and grass-leaved sweetflag extract remainder add the total medicinal material 6-10 times weight concentration of prescription, heating and refluxing extraction 1-3 time, each 1-2 hour, extract merges, leave standstill, filter, decompression filtrate recycling ethanol, be concentrated into the clear cream that relative density under 60 DEG C of conditions is 1.15 ~ 1.30;

E, the microcrystalline cellulose of clear cream and the heavy 30-40% of clear cream to be mixed, put drying under reduced pressure under 60 DEG C of-80 DEG C of conditions, get dry extract;

F, dry cream and cyclodextrin inclusion compound are ground into fine powder respectively, weigh 0.5% dolomol with the microcrystalline cellulose of the heavy 5-55% of clear cream and dry cream and mix, obtain fine powder, filled capsules.

The preparation method of compound silver Lingtong capsule specifically can in accordance with the following steps:

Formula: ginkgo leaf 333g, glossy ganoderma 333g, radix scrophulariae 278g, Radix Achyranthis Bidentatae 278g, rhizoma Gastrodiae 125g, grass-leaved sweetflag 167g.

Preparation method:

(1) pulverize: grass-leaved sweetflag is ground into particle, for subsequent use.

(2) CO ₂supercritical extract: get grass-leaved sweetflag particle in extraction kettle, CO ₂supercritical extract, wherein extracting pressure is 30Mpa, and extraction kettle temperature is 60 DEG C, and extraction time is 1.5 hours, obtains volatile oil and grass-leaved sweetflag extract remainder.

(3) inclusion essential oil: get the isopyknic ethanol of volatile oil and dissolve, obtain volatile oil ethanolic solution; Separately get the beta-schardinger dextrin-of volatile oil weight 4 times amount, grind well with the water of beta-schardinger dextrin-4 times amount, pour in colloid mill, slowly continuously drip above-mentioned volatile oil ethanolic solution, the inclusion 20 minutes of milling, inclines and, 0-4 DEG C of refrigeration leaves standstill more than 24 hours, filter, in 40 DEG C of vacuum drying, obtain volatile oil clathrate compound;

(4) alcohol extracting: it is the ethanol of 90% that glossy ganoderma, ginkgo leaf, Radix Achyranthis Bidentatae, radix scrophulariae and rhizoma Gastrodiae and grass-leaved sweetflag extract remainder add the total medicinal material of prescription 8 times of weight concentrations, heating and refluxing extraction twice, each 1.5 hours, extract merges, leave standstill, filter, decompression filtrate recycling ethanol, be concentrated into the clear cream that relative density under 60 DEG C of conditions is 1.15 ~ 1.30, measure dry cream rate.

(5) combination drying: mixed by the microcrystalline cellulose of clear cream and the heavy 30-40% of clear cream, puts drying under reduced pressure under vacuum drying chamber 60 DEG C of conditions, gets dry extract.

(6) pulverizing, mixing, capsule-filling: dry cream, volatile oil clathrate compound are put in Universalpulverizer and be ground into fine powder, weigh 0.5% dolomol with residue microcrystalline cellulose and dry cream and mix, obtain fine powder; Wherein, the total consumption of microcrystalline cellulose of step (five) and (six) and the part by weight of clear cream are 237.5:260.

(7) capsule-filling: get No. 0 hard shell capsules and fill, loading amount: 0.5g

This product is hard capsule, and content is pistac powder, and gas is fragrant, mildly bitter flavor.

The object of the invention is to realize in the following manner:

A quality determining method for compound silver Lingtong capsule, this detection method comprises discriminating and assay, and wherein content assaying method comprises the following steps:

A) compound silver Lingtong capsule content is got in the preparation of need testing solution, fine ground, gets 2.9 ~ 3.1g, add sherwood oil 25 ~ 35ml that boiling point is 60 ~ 90 DEG C, ultrasonic process 20 ~ 40 minutes, centrifugal 3 ~ 8 minutes, discard sherwood oil liquid, add ultrasonic 20 ~ 40 minutes of sherwood oil 25 ~ 35ml that boiling point is 60 ~ 90 DEG C again, centrifugal 3 ~ 8 minutes, discard sherwood oil liquid, add methyl alcohol 25 ~ 35ml, ultrasonic process 20 ~ 40 minutes, centrifugal 3 ~ 8 minutes, collects methanol solution; Add methyl alcohol 25 ~ 35ml again, ultrasonic process 20 ~ 40 minutes, centrifugal 3 ~ 8 minutes, collect methanol solution, recovered under reduced pressure methyl alcohol is to dry, residue adds 30 ~ 80 DEG C of warm water 15 ~ 25ml, add ethyl acetate jolting to extract 3 ~ 4 times, each 15 ~ 25ml, collect acetic acid ethyl fluid, recycling design is to dry, the gradation of residue 5ml methyl alcohol is transferred in 10ml measuring bottle, and add water 3.5 ~ 4.5ml, ultrasonic process 20 ~ 40 minutes, take out, let cool, add methyl alcohol to scale, shake up, use 0.45um filtering with microporous membrane, get the analysis of subsequent filtrate sample introduction.

B) ginkalide A reference substance is got in the preparation of reference substance solution, ginkolide B reference substance, ginkalide C reference substance, Bilobalide reference substance are appropriate, adding concentration is the mixed solution that 50% methyl alcohol makes each bilobalide-containing A0.16-0.2mg of every 1ml, ginkolide B 0.06-0.1mg, ginkalide C 0.08-0.12mg, Bilobalide 0.18-0.22mg respectively, to obtain final product.

C) determination method often plants reference substance accurate absorption reference substance solution 5 μ l, 10 μ l, 20 μ l respectively, need testing solution draws 10 ~ 20 μ l, injection liquid chromatography, measure, calculate the content of ginkalide A, ginkolide B, ginkalide C and Bilobalide with external standard two-point method logarithmic equation respectively, to obtain final product;

Chromatographic condition and system flexibility octadecylsilane chemically bonded silica are filling agent; Volume ratio is the methyl alcohol-tetrahydrofuran-water of 25:10:65 is mobile phase, and evaporation scatter detector detects, and number of theoretical plate calculates by Bilobalide peak and is not less than 3000.

Above-mentioned content assaying method preferably includes following steps:

A) compound silver Lingtong capsule content is got in the preparation of need testing solution, fine ground, gets 3g, add the sherwood oil 30ml that boiling point is 60 ~ 90 DEG C, ultrasonic process 30 minutes, centrifugal 5 minutes, discard sherwood oil liquid, add ultrasonic 30 minutes of the sherwood oil 30ml that boiling point is 60 ~ 90 DEG C again, centrifugal 5 minutes, discard sherwood oil liquid, add methyl alcohol 30ml, ultrasonic process 30 minutes, centrifugal 5 minutes, collects methanol solution; Add methyl alcohol 30ml again, ultrasonic process 30 minutes, centrifugal 5 minutes, collect methanol solution, recovered under reduced pressure methyl alcohol is to dry, and residue adds 30 ~ 80 DEG C of warm water 20ml, adds ethyl acetate jolting and extracts 3 times, each 20ml, merges and collects acetic acid ethyl fluid, and recycling design is to dry, the gradation of residue 5ml methyl alcohol is transferred in 10ml measuring bottle, and add water 4.5ml, ultrasonic process 30 minutes, take out, let cool, add methyl alcohol to scale, shake up, use 0.45um filtering with microporous membrane, get the analysis of subsequent filtrate sample introduction;

B) ginkalide A reference substance is got in the preparation of reference substance solution, ginkolide B reference substance, ginkalide C reference substance, Bilobalide reference substance are appropriate, adding concentration is the mixed solution that 50% methyl alcohol makes each bilobalide-containing A0.18mg of every 1ml, ginkolide B 0.08mg, ginkalide C 0.1mg, Bilobalide 0.2mg respectively, to obtain final product;

Chromatographic condition and system flexibility octadecylsilane chemically bonded silica are filling agent; Volume ratio is the methyl alcohol-tetrahydrofuran-water of 25:10:65 is mobile phase, and evaporation scatter detector detects.Number of theoretical plate calculates by Bilobalide peak and is not less than 3000.Compound silver Lingtong capsule composition containing ginkgo leaf is with the total amount of ginkalide A, ginkolide B, ginkalide C and Bilobalide, and every is no less than 0.44mg.

In above-mentioned discrimination method, comprise glossy ganoderma discriminating, radix scrophulariae is differentiated, grass-leaved sweetflag is differentiated, concrete steps are as follows:

A, with glossy ganoderma control medicinal material for contrast, thin layer condition is thin layer G plate, and employing volume ratio is 10 ~ 14:10 ~ 14:0.5 toluene-ethyl acetate-acetic acid mixed solution is developping agent, and the ethanol solution of sulfuric acid of 10% is developer, and 105 DEG C are heated to spot development;

B, with radix scrophulariae control medicinal material for contrast, then get harpagoside reference substance, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast; Thin layer condition is silica gel g thin-layer plate, and employing volume ratio is lower floor's solution of the chloroform-methanol-water mixed solution of 10 ~ 14:3 ~ 5:1 is developping agent, and 5% vanillin-sulfuric acid ethanolic solution is developer, and 105 DEG C are heated to spot development;

C, with grass-leaved sweetflag control medicinal material for contrast, thin layer condition is silica gel g thin-layer plate, and employing volume ratio is 3 ~ 5:1, and boiling point is the sherwood oil of 60 ~ 90 DEG C and ethyl acetate mixture is developping agent, places 1 hour, inspects under putting 365nm ultraviolet lamp.

In above-mentioned discrimination method, comprise glossy ganoderma discriminating, radix scrophulariae is differentiated, grass-leaved sweetflag is differentiated, preferred concrete steps are as follows:

A, with glossy ganoderma control medicinal material for contrast, thin layer condition is thin layer G plate, and adopt volume ratio be 12:12:0.5 toluene-ethyl acetate-acetic acid mixed solution to be developping agent, the ethanol solution of sulfuric acid of 10% is developer, and 105 DEG C are heated to spot development;

B, with radix scrophulariae control medicinal material for contrast, then get harpagoside reference substance, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast; Thin layer condition is silica gel g thin-layer plate, and employing volume ratio is lower floor's solution of the chloroform-methanol-water mixed solution of 12:4:1 is developping agent, and 5% vanillin-sulfuric acid ethanolic solution is developer, and 105 DEG C are heated to spot development;

C, with grass-leaved sweetflag control medicinal material for contrast, thin layer condition is silica gel g thin-layer plate, and employing volume ratio is 4:1, and boiling point is the sherwood oil of 60 ~ 90 DEG C and ethyl acetate mixture is developping agent, places 1 hour, inspects under putting 365nm ultraviolet lamp.

Glossy ganoderma discrimination method specifically comprises the following steps:

Get compound silver Lingtong capsule content 3g, add chloroform backflow twice, each 50ml, first time backflow 3 hours, second time backflow 1 hour, merging filtrate, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Separately get glossy ganoderma control medicinal material 0.5g, be made in the same way of control medicinal material solution.According to thin-layered chromatography, drawing each 4 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, is that the toluene-ethyl acetate-acetic acid mixed solution of 12:12:0.5 is developping agent with volume ratio, launch, take out, dry, spraying with concentration is 10% ethanol solution of sulfuric acid, spot development is heated to clear at 105 DEG C, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

Radix scrophulariae discrimination method specifically comprises the following steps:

Get compound silver Lingtong capsule content 6g, add methyl alcohol 25ml, soak 1 hour, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, the residue 25ml that adds water makes dissolving, with water saturated normal butyl alcohol shaking out 2 times, each 30ml, merges normal butyl alcohol liquid, evaporate to dryness, residue adds methyl alcohol 5ml makes dissolving, as need testing solution; Separately get radix scrophulariae control medicinal material 2g, be made in the same way of control medicinal material solution.Get harpagoside reference substance again, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.According to thin-layered chromatography, drawing each 4 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, is that lower floor's solution of the chloroform-methanol-water mixed solution of 12:4:1 is developping agent with volume ratio, launch, take out, dry, spraying with concentration is 5% vanillin-sulfuric acid ethanolic solution, spot development is heated to clear at 105 DEG C, in test sample chromatogram, on the position corresponding with control medicinal material chromatogram to reference substance chromatogram, the spot of aobvious same color.

Grass-leaved sweetflag discrimination method specifically comprises the following steps:

Get compound silver Lingtong capsule content 1g, add the sherwood oil 20ml that boiling point is 60 ~ 90 DEG C, ultrasonic process 20 minutes, filter, filtrate evaporate to dryness, it is that the sherwood oil 1ml of 60 ~ 90 DEG C makes dissolving, as need testing solution that residue adds boiling point; Separately get grass-leaved sweetflag control medicinal material 0.2g, be made in the same way of control medicinal material solution.According to thin-layered chromatography, drawing each 2 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, is that the petroleum ether-ethyl acetate mixed solution of 60 ~ 90 DEG C is for developping agent with volume ratio 4:1 boiling point, launch, take out, dry, place 1 hour, inspect under putting 365nm ultraviolet lamp, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the fluorescence spot of aobvious same color.

The preparation of negative control solution in discrimination process: get not containing other medicinal materials of medicinal material to be identified by prescription, extract by preparation technology, concentrate, obtain negative control medicinal extract.Get the medicinal extract be equivalent to contained by a certain amount of content of capsule, by need testing solution preparation method process, obtain negative control solution.

Beneficial effect of the present invention compared with the prior art: method of quality control separation efficiency of the present invention is high, good stability, analysis speed are fast, the method controlling compound silver Lingtong capsule reliable in quality, for the quality control of compound silver Lingtong capsule finished product establishes an accurate feasible control criterion.

The present invention is specifically addressed (in test, compound silver Lingtong capsule all prepares according to embodiment 1 method) by following test example.

Test example 1. content assaying method parameter selection experiments

1, instrument and reagent:

Agilent1200 series of high efficiency liquid chromatograph G1310A etc. spend chromatogram pump, Varian380 evaporative light-scattering detector (ELSD).

Reference substance: ginkalide A (lot number: 110862-200608), ginkolide B (lot number: 110863-200508), ginkalide C (lot number: 110864-200906), Bilobalide (110865-200605) are purchased from Nat'l Pharmaceutical & Biological Products Control Institute.Compound silver Lingtong capsule, prepares according to embodiment 1 method.

2, chromatography condition

2.1 mobile phase

The assay method of ginkgolides in Primary Reference Chinese Pharmacopoeia 2010 editions ginkgo leaf medicinal materials when selecting chromatogram flow phase in process of the test, mobile phase used is methyl alcohol-tetrahydrofuran-water (25:10:65), on this basis, investigate the separation case after adjustment mobile phase ratio, in table 1.

Table 1 changes the impact that mobile phase ratio is separated ginkgolides

The separation impact of ratio on ginkgolides of visible a small amount of change methyl alcohol-tetrahydrofuran-water is little, but point defection wherein between Bilobalide and ginkalide A is affected, especially when sample separation, impact is larger, therefore the separation case of comprehensive retention time and sample, still selects methyl alcohol-tetrahydrofuran-water (25:10:65) as mobile phase.

2.2 chromatographic column

Different brands, different length, varigrained chromatographic column are investigated to the impact of ginkgolides separating effect, in table 2.

The different chromatographic column sample separation of table 2 compares

*: column temperature 35 DEG C, flow velocity 1ml/min.

*: chromatographic column is shorter, stationary-phase particle size is thin, flow rate of mobile phase is 0.5ml/min.

Ginkgolide A. B. C and Bilobalide all can separate by the chromatographic column different as seen by table 2, but it is different to the separating effect of sample, consider retention time and separation case, selection Shim-PackODS (150 × 4.6mm, 5m) as follow-up study chromatographic column.

2.3 column temperature

Be under the condition of methyl alcohol-tetrahydrofuran-water (25:10:65), flow velocity 1ml/min at mobile phase, adopt column oven to control column temperature, investigated the impact of different temperatures on separating effect, the results are shown in Table 3.

Table 3 different temperatures is on the impact of separating effect

The visible rising along with temperature, the retention time of each component all shortens, but little on the impact be separated.

2.4ELSD testing conditions is optimized

During use ELSD detection sample, its sensitivity is by atomization temperature, evaporating temperature and N ₂the impact of flow velocity, investigate the methanol solution of the ginkolide B minimum using concentration in the test of ELSD detection sensitivity, response is less as the direct Injection Detector of sample, investigate the influence factor of relative detector response, record peak height, the condition time the highest with peak height is for the best.Experimental result is in table 4 (wherein peak height is the mean value of 2 times or 3 times tests).

Table 4ELSD testing conditions is optimized

From table 10, under same evaporating temperature (90 DEG C), atomization temperature raises, and response reduces; Under identical atomization temperature (60 DEG C), evaporating temperature raises, and response becomes large; Time atomization temperature identical with evaporating temperature (70 DEG C and 90 DEG C), increase flow rate of carrier gas, response diminishes.The use of integrated instrument and larger response requirement, the testing conditions selecting ELSD is atomization temperature 60 DEG C, evaporating temperature 110 DEG C, flow rate of carrier gas 1.0SLM.

3, chromatographic condition is determined

Chromatographic column: Shim-packVP-ODS150mm × 4.6 μm ID, 5 μm

Mobile phase: methyl alcohol-tetrahydrofuran-water (25:10:65), flow velocity 1ml/min

Column temperature: 35 DEG C

Evaporative light-scattering detector detects, testing conditions: evaporating temperature 110 DEG C, atomization temperature 60 DEG C, N ₂flow velocity 1.0SLM

4, sample-pretreating method is selected

4.1 extracting method

With reference to the assay method of ginkgolides in gingko leaf preparation in the assay method of ginkgolides in Chinese Pharmacopoeia 2010 editions ginkgo leaf medicinal materials and document in test, compare soxhlet extraction and ultrasonic (350W, 50kHz) extraction method extracts the efficiency of the ginkgolides in sample, has investigated the method for subsequent processing of two kinds of extracts: alumina column method and liquid-liquid extraction method simultaneously.

1) soxhlet extraction:

Get compound silver Lingtong capsule content 4g, accurately weighed, put in apparatus,Soxhlet's, add sherwood oil (30 ~ 60 DEG C) refluxing extraction 1 hour in 70 DEG C of water-baths, discard sherwood oil (30 ~ 60 DEG C) liquid, the dregs of a decoction and filtration paper cylinder wave most sherwood oil, be placed in 60 DEG C of baking ovens to dry, add methanol eddy again and extract 6 hours, extract evaporate to dryness, residue adds methyl alcohol makes dissolving, be transferred in 10ml measuring bottle, ultrasonic (350W, 50kHz) process 30 minutes, take out, let cool, add methyl alcohol to scale, shake up, leave standstill, precision measures supernatant 5ml, add acidic alumina column (200 ~ 300 orders, 3g, internal diameter is 1cm, post is filled with methanol wet) on, with methyl alcohol 25ml wash-out, collect eluent, recycling design is to dry, residue methyl alcohol 5ml gradation is transferred in 10ml measuring bottle, add water 4.5ml, ultrasonic (350W, 50kHz) process 30 minutes, take out, let cool, add methyl alcohol to scale, shake up, let cool, with 0.45 μm of filtering with microporous membrane, get the analysis of subsequent filtrate sample introduction.

2) ultrasonic extraction:

Get compound silver Lingtong capsule content 4g, accurately weighed, put in 30ml glass centrifuge tube, add sherwood oil (60 ~ 90 DEG C) 30ml, ultrasonic (350W, 50kHz) process 30 minutes, centrifugal 5 minutes (2000 revs/min), discard sherwood oil liquid, add the ultrasonic (350W of sherwood oil (60 ~ 90 DEG C) 30ml again, 50kHz) 30 minutes, centrifugal 5 minutes (2000 revs/min), discarded sherwood oil liquid.In centrifuge tube, add methyl alcohol 30ml, ultrasonic (350W, 50kHz) processes 30 minutes, centrifugal 5 minutes (2000 revs/min), collects methanol solution, methyl alcohol 30ml is added again in centrifuge tube, ultrasonic (350W, 50kHz) process 30 minutes, centrifugal 5 minutes (2000 revs/min), merge methanol solution, reclaim methyl alcohol to dry, residue adds warm water 20ml, 3 times are extracted with ethyl acetate jolting, each 20ml, combined ethyl acetate liquid, recycling design is to dry, residue methyl alcohol 5ml gradation is transferred in 10ml measuring bottle, add water 4.5ml, ultrasonic (350W, 50kHz) process 30 minutes, take out, let cool, add methyl alcohol to scale, shake up, let cool, with 0.45 μm of filtering with microporous membrane, get the analysis of subsequent filtrate sample introduction.

By above-mentioned two kinds of method process compounds silver Lingtong capsule sample, analyze through HPLC, soxhlet extraction gained chromatographic peak area is starkly lower than ultrasonic extraction, and ultrasonic extraction required time is short, and soxhlet extraction is consuming time, process loaded down with trivial details, therefore select ultrasonic extraction as sample extraction method.Show that extraction into ethyl acetate method is better than alumina column process to the test findings of the post-processing approach of the sample solution after extraction, therefore the processing mode of sample selects ultrasonic extraction-extraction into ethyl acetate.

4.2 extraction time

Sample is after sherwood oil ultrasonic extraction removing liposoluble constituent, and the extraction time adding methyl alcohol may affect the extraction completeness of ginkgolides.This test is after sample adds the ultrasonic extraction of methyl alcohol twice, and third time adds methyl alcohol 30ml, after ultrasonic process, centrifugal, gets supernatant and volatilizes, and residue adds 0.5ml methyl alcohol and dissolves, and with 0.45 μm of filtering with microporous membrane, gets the analysis of subsequent filtrate sample introduction.Result chromatogram is without the signal of ginkgolides to be measured, and therefore methyl alcohol extracts twice basic extraction completely.

5, the preparation of solution

The preparation of 5.1 reference substance solution

Get ginkalide A reference substance, ginkolide B reference substance, ginkalide C reference substance, Bilobalide reference substance are appropriate, add the mixed solution that 50% methyl alcohol makes each bilobalide-containing A0.18mg of every 1ml, ginkolide B 0.08mg, ginkalide C 0.10mg, Bilobalide 0.20mg respectively, to obtain final product.

The preparation of 5.2 need testing solutions

Get capsule 's content powder 3g, accurately weighed, put in 30ml glass centrifuge tube, add sherwood oil (60 ~ 90 DEG C) 30ml, ultrasonic (350W, 50kHz) process 30 minutes, centrifugal 5 minutes (2000 revs/min), discard sherwood oil liquid, add the ultrasonic (350W of sherwood oil (60 ~ 90 DEG C) 30ml again, 50kHz) 30 minutes, centrifugal 5 minutes (2000 revs/min), discarded sherwood oil liquid.In centrifuge tube, add methyl alcohol 30ml, ultrasonic (350W, 50kHz) processes 30 minutes, centrifugal 5 minutes (2000 revs/min), collects methanol solution; In centrifuge tube, add methyl alcohol 30ml again, ultrasonic (350W, 50kHz) processes 30 minutes, centrifugal 5 minutes (2000 revs/min), merge methanol solution, recovered under reduced pressure methyl alcohol is to dry, and residue adds warm water 20ml, 3 times are extracted with ethyl acetate jolting, each 20ml, combined ethyl acetate liquid, recycling design is to dry, the gradation of residue 5ml methyl alcohol is transferred in 10ml measuring bottle, add water 4.5ml, and ultrasonic (350W, 50kHz) processes 30 minutes, take out, let cool, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, get the analysis of subsequent filtrate sample introduction.

6, determination method

Accurate absorption reference substance solution 5 μ l, 10 μ l, 20 μ l respectively, need testing solution 10 ~ 20 μ l, injection liquid chromatography, measure, calculate the content of ginkalide A, ginkolide B, ginkalide C and Bilobalide with external standard two-point method logarithmic equation respectively, calculate total lactones content, to obtain final product.

7, methodological study

7.1 specificities are investigated

Take other medicinal materials of not composition containing ginkgo leaf by prescription, prepare negative control solution by the preparation method of 7.5.2 need testing solution, with sample introduction under sample identical chromatographic conditions, record chromatogram show that in prescription, other medicinal materials do not disturb the mensuration of ginkgolides.

7.2 typical curve

1) preparation of stock solution: take ginkalide A, ginkolide B, ginkalide C and Bilobalide appropriate, add the mixed solution that 50% methyl alcohol makes each bilobalide-containing A0.9mg of every 1ml, ginkolide B 0.4mg, ginkalide C 0.5mg, Bilobalide 1mg respectively, as stock solution.

2) precision pipettes in stock solution 0.5ml to 10ml measuring bottle, adds 50% methyl alcohol to scale, in contrast product solution a.Precision pipettes in stock solution 1ml to 5ml measuring bottle, adds 50% methyl alcohol to scale, in contrast product solution b.Precision pipettes in stock solution 1ml to 2ml measuring bottle, adds 50% methyl alcohol to scale, in contrast product solution c.Accurate absorption reference substance solution a5 μ l, 10 μ l, 20 μ l and reference substance solution b10 μ l, 20 μ l, and reference substance solution c12 μ l, injection liquid chromatography, record chromatogram and peak area, chromatogram is shown in accompanying drawing, return with the common logarithm of the common logarithm of each component sample size (μ g) and peak area, obtain the regression curve of each component.Visible ginkalide C exists scope is interior, related coefficient γ=0.9995, Bilobalide within the scope of 0.256 ~ 6.132 μ g, related coefficient γ=0.9998, ginkalide A within the scope of 0.232 ~ 5.580 μ g, related coefficient γ=0.9999, ginkolide B is within the scope of 0.105 ~ 2.514 μ g, related coefficient γ=0.9991, sample size (μ g) and the peak area of component to be measured have good double-log linear relationship.

7.3 solution stability testing

Extracting sample solution, respectively at 0,2,4,8,12 hours sample introductions, the RSD of each component peaks area is in table 5, the peak area RSD of the single component of visible ginkgolides is larger, but calculating total lactones content RSD through typical curve is 3.0%, in interpret sample solution, total lactones content is better at 8 hours internal stabilities, and the sample solution of preparation measured in 8 hours.

Table 5 sample solution stability experiment result

7.4 sample introduction precision tests

Reference substance solution a20 μ l in the test of accurate absorption typical curve, injection liquid chromatography, altogether sample introduction 6 times, record chromatogram and peak area, the peak area RSD of each component is in table 6, and visible sample introduction precision is better.

Table 6 sample introduction Precision test result

7.5 replica test

Get silver-colored Lingtong capsule (lot number: 20120401) content powder 3g, totally six parts, by preparation method's parallel processing of need testing solution under 7.5.2 item, the each solution 20 μ l of accurate absorption, injection liquid chromatography, record chromatogram and peak area, in calculation sample, the content of ginkalide A, ginkolide B, ginkalide C and Bilobalide and the content of total lactones, the results are shown in Table 7.The RSD of total lactones content is 1.7%, the method repeatability is described better.

Table 7 replica test result

7.6 recovery test

Get silver-colored Lingtong capsule (lot number: 20120401) content powder 1.5g, totally six parts, put in 30ml glass centrifuge tube, to often propping up in centrifuge tube the ginkalide A, ginkolide B, ginkalide C and the Bilobalide that add and be equivalent to contained by 1.5g sample, by preparation method's parallel processing of need testing solution under 7.5.2 item, each solution 20 μ l of accurate absorption, injection liquid chromatography, record chromatogram and peak area, the average recovery of calculation and analysis methods, in table 8.Result shows that this law recovery is between 99.2 ~ 103.1%, and the average recovery rate of total lactones is 101.5%, RSD is 1.5%.

Table 8 recovery test result

7.7 medicinal material assays

Get Ginkgo Leaf 1.5g, accurately weighed, process by sample treatment under 7.5.2 item, extracting sample solution 10-20 μ l sample introduction, get reference substance solution sample introduction, record chromatogram and peak area, chromatogram is shown in accompanying drawing simultaneously, calculate the content of total lactones in ginkgo leaf medicinal material by double-log equation, the results are shown in Table 9.

Total Terpene Lactones assay result in table 9 three batches of ginkgo leaf medicinal materials

A: assay is undertaken by under Chinese Pharmacopoeia 2010 editions ginkgo leaf items, sample size 15 μ l, each sample feeding once.

B: assay is undertaken by under Chinese Pharmacopoeia 2010 editions ginkgo leaf items, sample size 15 μ l, each sample feeding twice, for the preparation of test agent in 20120401,20120402,20120403.

C: assay this product content assaying method carries out, sample size 10 μ l, each sample feeding twice, for the preparation of test agent in 20130601,20130602,20130603.

7.8 sample sizes measure

Get silver-colored Lingtong capsule (lot number: 20120402,20120403,20130601,20130602,20130603) content powder 3g, each lot number gets two parts, by preparation method's parallel processing of need testing solution under 7.5.2 item, the each solution 20 μ l of accurate absorption, injection liquid chromatography, record chromatogram and peak area, in calculation sample, the content of ginkalide A, ginkolide B, ginkalide C and Bilobalide and the content of total lactones, the results are shown in Table 10.

Total Terpene Lactones assay result in table 10 six batch sample

From table 10, in six batches, in test agent, the content of ginkalide A, ginkolide B, ginkalide C and Bilobalide is all lower than 0.05%, and thus independent lactone is not suitable for the assay index as preparation.Specify under assay item with reference to ginkgo leaf medicinal material in Chinese Pharmacopoeia 2010 editions, calculate the content of total Terpene Lactones in this product, result is all greater than 0.1%, therefore can using the assay index of the mensuration of total Terpene Lactones as silver-colored Lingtong capsule.

The rate of transform calculating 20120401,20120402,20,120,403 3 batch sample ginkgolides according to the ginkgo biloba leaf total terpene lactone content (0.260%) used during pilot scale is respectively 59.5%, 64.7%, 68.1%, the rate of transform that in like manner can calculate the ginkgolides of 20130601,20130602,20,130,603 3 batch samples is respectively 62.2%, 60.7%, 59.8%, therefore the average content of test agent is 0.110% in above-mentioned six batches, and mean transferred rate is 62.5%.According to average content, and consider floating of crude drug content, therefore sample average content floated downward 20%, namely in every compound silver curing capsule composition containing ginkgo total lactones 0.44mg as content limit.So finished product content limit is defined as: this product every composition containing ginkgo leaf should be less than 0.44mg in total Terpene Lactones.

Formulate corresponding intermediate quality standard as inner quality standard according to pilot-scale experiment, the results are shown in Table 11.

Table 11. intermediate quality standard (internal control)

Test example 2: the selection of glossy ganoderma discrimination method condition in compound silver Lingtong capsule:

Test method one: get capsule 's content 3g, adds ethanol 30ml, adds hot reflux 30 minutes, and filter, filtrate evaporate to dryness, residue adds methyl alcohol 2ml makes dissolving, as need testing solution.Separately get glossy ganoderma control medicinal material 2g, be made in the same way of control medicinal material solution.Test according to thin-layered chromatography (Chinese Pharmacopoeia 2010 editions annex VIB), draw each 4 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with the upper solution of sherwood oil (60 ~ 90 DEG C)-ethyl formate-formic acid (15:5:1) for developping agent, launch, take out, dry, inspect under putting uviol lamp (365nm), in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of same color should be shown.

Test method two: get capsule 's content 3g, adds chloroform backflow twice, each 50ml, first time backflow 3 hours, and second time backflow 1 hour, merging filtrate, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Separately get glossy ganoderma control medicinal material 0.5g, be made in the same way of control medicinal material solution.Separately get not containing other medicinal materials of glossy ganoderma by prescription, extract according to preparation technology, concentrate, by need testing solution, preparation method makes negative control solution.Test according to thin-layered chromatography (Chinese Pharmacopoeia 2010 editions annex VIB), draw each 4 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-acetic acid (12:12:0.5) for developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of same color should be shown.

Method one is the discrimination method of glossy ganoderma medicinal material in Chinese Pharmacopoeia (2010 editions), and thin-layer chromatogram is shown in Fig. 1, and under finding this condition in process of the test, this product thin-layer chromatogram is under 365nm fluorescence, and fluorescence spot is not obvious, but control medicinal material has significant spot; Under 254nm uviol lamp, spot blackening position is consistent with under 365nm, and therefore the method can not be used for differentiating glossy ganoderma.Method one does not detect fluorescence spot may be relevant with preparation process and processing procedure.

Main containing polysaccharide and ganodenic acid acid in bibliographical information glossy ganoderma, wherein ganodenic acid acid compounds is its characteristic component.The present invention selects to obtain the glossy ganoderma medicinal material that test method two is differentiated in prescription, thin-layer chromatogram is shown in Fig. 2, under 254nm uviol lamp, control medicinal material and sample solution identical bits are equipped with blackening, through sulfuric acid-ethanol colour developing, spot is in significantly pink, and negative control is noiseless, can be used as the discrimination method of glossy ganoderma in this product.Test point sample amount to the impact be separated, see Fig. 3, namely visible point sample 4 μ l can be observed obvious spot, and does not affect separation simultaneously.Therefore system of selection two is as the discrimination method of glossy ganoderma in silver-colored curing capsule, and in three batch samples, Fig. 3 is shown in the discriminating of glossy ganoderma.

Test example 3: the selection of radix scrophulariae discrimination method condition in compound silver Lingtong capsule:

Test method: get capsule 's content 6g, adds methyl alcohol 25ml, soaks 1 hour, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, the residue 25ml that adds water makes dissolving, with water saturated normal butyl alcohol shaking out 2 times, each 30ml, merges normal butyl alcohol liquid, evaporate to dryness, residue adds methyl alcohol 5ml makes dissolving, as need testing solution.Separately get radix scrophulariae control medicinal material 2g, be made in the same way of control medicinal material solution.Get harpagoside reference substance again, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.Separately get not containing other medicinal materials of radix scrophulariae by prescription, extract according to preparation technology, concentrate, by need testing solution, preparation method makes negative control solution.Test according to thin-layered chromatography (Chinese Pharmacopoeia 2010 editions annex VIB), draw each 4 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with lower floor's solution of chloroform-methanol-water (12:4:1) for developping agent, launch, take out, dry, spray, with 5% vanillin-sulfuric acid ethanolic solution, is heated to spot development at 105 DEG C clear.In test sample chromatogram, on the position corresponding with control medicinal material chromatogram to reference substance chromatogram, the spot of aobvious same color.

The condition test thin-layer chromatogram that radix scrophulariae medicinal material is differentiated is shown in Fig. 4.Result shows that negative control is noiseless.Under directly putting 254nm ultraviolet light after expansion, in sample, spot is not obvious, sprays with vanillin-sulfuric acid ethanolic solution, can see the obvious spot consistent with reference substance 105 DEG C of heating, therefore can be used as the discrimination method of radix scrophulariae in compound silver Lingtong capsule, in three batch samples, Fig. 5 is shown in the discriminating of radix scrophulariae.

Test example 4: the selection of grass-leaved sweetflag discrimination method condition in compound silver Lingtong capsule:

Test method: get capsule 's content powder 1g, adds sherwood oil (60 ~ 90 DEG C) 20ml, ultrasonic process 20 minutes, and filter, filtrate evaporate to dryness, residue adds sherwood oil (60 ~ 90 DEG C) 1ml makes dissolving, as need testing solution.Separately get grass-leaved sweetflag control medicinal material 0.2g, be made in the same way of control medicinal material solution.Separately get not containing other medicinal materials of grass-leaved sweetflag by prescription, extract according to preparation technology, concentrate, by need testing solution, preparation method makes negative control solution.Test according to thin-layered chromatography (Chinese Pharmacopoeia 2010 editions annex VIB), draw each 2 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (60 ~ 90 DEG C)-ethyl acetate (4:1) for developping agent, launch, take out, dry, place 1 hour, inspect under putting ultraviolet lamp (365nm).In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the fluorescence spot of aobvious same color.

In silver curing capsule, ultrasonic process is selected in the discriminating of grass-leaved sweetflag, shorten extraction time, decrease medicinal material consumption, launch by above-mentioned test method, thin-layer chromatogram is shown in Fig. 6, and under visible 365nm ultraviolet light, sample has fluorescence spot on the position corresponding to control medicinal material, namely point sample 2 μ l can be observed obvious fluorescence spot, and negative control is noiseless, therefore can be used as the discrimination method of grass-leaved sweetflag, in three batches of silver-colored Lingtong capsule samples, Fig. 7 is shown in the discriminating of grass-leaved sweetflag.

All the other, measure moisture in capsule 's content according to " Chinese Pharmacopoeia " version in a 2010 middle aquametry (annex IXH).Three batch sample measurement results are all less than 9%, conform with the regulations.Carry out content uniformity by " Chinese Pharmacopoeia " 2010 editions annex, disintegration time limited conforms with the regulations; Carry out microbial limit according to " Chinese Pharmacopoeia " version in a 2000 middle microbial decolorization (annex XIIIC), result shows that this product microbial limit all conforms with the regulations.

Accompanying drawing explanation

The thin-layer chromatogram of glossy ganoderma in Fig. 1 test method one compound silver Lingtong capsule.

Inspect figure under Zuo Tu: 365nm fluorescence, right figure is: inspect figure under 254nm uviol lamp.

On figure, sample spot is respectively control medicinal material, sample solution 2 μ l, sample solution 4 μ l from left to right.

Glossy ganoderma thin-layer chromatogram in Fig. 2 test method two compound silver Lingtong capsule.

Figure is inspected, right figure: sulfuric acid-ethanol colour developing figure under Zuo Tu: 254nm uviol lamp.

Sample spot is respectively control medicinal material, sample solution, negative control solution from left to right.

In Fig. 3 compound silver Lingtong capsule, glossy ganoderma sample solution point sample amount investigates figure.

Wherein, the discriminating figure of left figure to be specificity investigation figure, right figure be three batch samples;

The discrimination condition test of radix scrophulariae in Fig. 4 compound silver Lingtong capsule.

Sample spot is from left to right: 1 is reference substance, and 2 is control medicinal material, and 3-6 is respectively sample solution 1 μ l, 2 μ l, 4 μ l, 6 μ l, and 7 is negative control;

The discriminating of radix scrophulariae in Fig. 5 tri-batches of compound silver Lingtong capsules.

Sample spot is from left to right: 1 is reference substance, and 2 is control medicinal material, and all the other are three batch samples.

In Fig. 6 silver curing capsule, the discrimination condition test of grass-leaved sweetflag is (left: 254nm, right: 365nm).

Sample spot is from left to right: 1 is control medicinal material, and 2-5 is respectively the molten 2 μ l of sample, 2 μ l, 3 μ l, 4 μ l, and 6 is negative control.

The discriminating of grass-leaved sweetflag in Fig. 7 tri-batches of compound silver Lingtong capsules.

Sample spot is from left to right: 1 is control medicinal material, and all the other are three batch samples.

Embodiment

Embodiment 1

Formula:

Ginkgo leaf 333g, glossy ganoderma 333g, radix scrophulariae 278g, Radix Achyranthis Bidentatae 278g, rhizoma Gastrodiae 125g, grass-leaved sweetflag 167g.

Preparation method:

Embodiment 2

The content assaying method of the compound silver Lingtong capsule prepared according to embodiment 1 method:

A) this product content is got in the preparation of need testing solution, fine ground, accurately weighed 3g, put in 30ml glass centrifuge tube, add sherwood oil (boiling point is 60 ~ 90 DEG C) 30ml, ultrasonic (350W, under 50kHz condition) process 30 minutes, centrifugal 5 minutes (under 2000 revs/min of conditions), discard sherwood oil liquid, then add sherwood oil (boiling point is 60 ~ 90 DEG C) 30ml ultrasonic (under 350W, 50kHz condition) 30 minutes, centrifugal 5 minutes (under 2000 revs/min of conditions), discard sherwood oil liquid.In centrifuge tube, add methyl alcohol 30ml, ultrasonic (under 350W, 50kHz condition) process 30 minutes, centrifugal 5 minutes (under 2000 revs/min of conditions), collect methanol solution, methyl alcohol 30ml is added again in centrifuge tube, ultrasonic (350W, 50kHz) process 30 minutes, centrifugal 5 minutes (under 2000 revs/min of conditions), collect methanol solution, recovered under reduced pressure methyl alcohol is to dry, residue adds 30 ~ 80 DEG C of warm water 20ml, 3 times are extracted with ethyl acetate jolting, each 20ml, collect acetic acid ethyl fluid, recycling design is to dry, the gradation of residue 5ml methyl alcohol is transferred in 10ml measuring bottle, add water 4.5ml, ultrasonic (350W, under 50kHz condition) process 30 minutes, take out, let cool, add methyl alcohol to scale, shake up, with 0.45 μm of filtering with microporous membrane, get the analysis of subsequent filtrate sample introduction.

B) ginkalide A reference substance is got in the preparation of reference substance solution, ginkolide B reference substance, ginkalide C reference substance, Bilobalide reference substance are appropriate, adding concentration is the mixed solution that 50% methyl alcohol makes each bilobalide-containing A0.18mg of every 1ml, ginkolide B 0.08mg, ginkalide C 0.10mg, Bilobalide 0.20mg respectively, to obtain final product.

C) determination method often plants reference substance accurate absorption reference substance solution 5 μ l, 10 μ l, 20 μ l respectively, need testing solution 10 ~ 20 μ l, injection liquid chromatography, measure, calculate the content of ginkalide A, ginkolide B, ginkalide C and Bilobalide with external standard two-point method logarithmic equation respectively, to obtain final product;

Chromatographic condition and system flexibility octadecylsilane chemically bonded silica are filling agent; Methyl alcohol-tetrahydrofuran-water (25:10:65), evaporation scatter detector detects.Number of theoretical plate calculates should be not less than 3000 by Bilobalide peak.

Compound silver Lingtong capsule composition containing ginkgo leaf is with ginkalide A (C ₂₀h ₂₄o ₉), ginkolide B (C ₂₀h ₂₄o ₁₀), ginkalide C (C ₂₀h ₂₄o ₁₁) and Bilobalide (C ₁₅h ₁₈o ₈) total amount, every is no less than 0.44mg.

The compound silver Lingtong capsule prepared according to embodiment 1 method carries out glossy ganoderma discriminating, radix scrophulariae discriminating, grass-leaved sweetflag discriminating,

Concrete steps are as follows:

A, get this product content 3g, add chloroform backflow twice, each 50ml, first time backflow 3 hours, second time backflow 1 hour, merging filtrate, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Separately get glossy ganoderma control medicinal material 0.5g, be made in the same way of control medicinal material solution.Test according to thin-layered chromatography " Chinese Pharmacopoeia " 2010 editions (annex VIB), draw each 4 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-acetic acid (12:12:0.5) for developping agent, launch, take out, dry, spray, with 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear.In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of same color should be shown.

B, get this product content 6g, add methyl alcohol 25ml, soak 1 hour, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, the residue 25ml that adds water makes dissolving, with water saturated normal butyl alcohol shaking out 2 times, each 30ml, merge normal butyl alcohol liquid, evaporate to dryness, residue adds methyl alcohol 5ml makes dissolving, as need testing solution.Separately get radix scrophulariae control medicinal material 2g, be made in the same way of control medicinal material solution.Get harpagoside reference substance again, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.Test according to thin-layered chromatography " Chinese Pharmacopoeia " 2010 editions (annex VIB), draw each 4 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with lower floor's solution of chloroform-methanol-water (12:4:1) for developping agent, launch, take out, dry, spray, with 5% vanillin-sulfuric acid ethanolic solution, is heated to spot development at 105 DEG C clear.In test sample chromatogram, on the position corresponding with control medicinal material chromatogram to reference substance chromatogram, the spot of aobvious same color.

C, get this product content 1g, add sherwood oil (boiling point is 60 ~ 90 DEG C) 20ml, ultrasonic process 20 minutes, filter, filtrate evaporate to dryness, residue adds sherwood oil (boiling point is 60 ~ 90 DEG C) 1ml makes dissolving, as need testing solution.Separately get grass-leaved sweetflag control medicinal material 0.2g, be made in the same way of control medicinal material solution.Test according to thin-layered chromatography " Chinese Pharmacopoeia " 2010 editions (annex VIB), draw each 2 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with sherwood oil (boiling point is 60 ~ 90 DEG C)-ethyl acetate (4:1) for developping agent, launch, take out, dry, place 1 hour, inspect under putting ultraviolet lamp (365nm).In test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the fluorescence spot of aobvious same color.

List of references

[1] pharmacopoeia commission of Ministry of Health of the People's Republic of China Pharmacopoeia of People's Republic of China (2010) version one.

Claims

1. a quality determining method for compound silver Lingtong capsule, it is characterized in that this detection method comprises discriminating and assay, wherein content assaying method comprises the following steps:

Chromatographic condition and system flexibility octadecylsilane chemically bonded silica are filling agent; Volume ratio is the methyl alcohol-tetrahydrofuran-water of 25:10:65 is mobile phase, and evaporation scatter detector detects, and number of theoretical plate calculates should be not less than 3000 by Bilobalide peak.

2. the quality determining method of compound silver Lingtong capsule according to claim 1, is characterized in that described content assaying method comprises the following steps:

A) compound silver Lingtong capsule content is got in the preparation of need testing solution, fine ground, gets 3g, add the sherwood oil 30ml that boiling point is 60 ~ 90 DEG C, ultrasonic process 30 minutes, centrifugal 5 minutes, discard sherwood oil liquid, add ultrasonic 30 minutes of the sherwood oil 30ml that boiling point is 60 ~ 90 DEG C again, centrifugal 5 minutes, discard sherwood oil liquid, add methyl alcohol 30ml, ultrasonic process 30 minutes, centrifugal 5 minutes, collects methanol solution; Add methyl alcohol 30ml again, ultrasonic process 30 minutes, centrifugal 5 minutes, collect methanol solution, recovered under reduced pressure methyl alcohol is to dry, and residue adds warm water 20ml, extracts 3 times with ethyl acetate jolting, each 20ml, collects acetic acid ethyl fluid, and recycling design is to dry, the gradation of residue 5ml methyl alcohol is transferred in 10ml measuring bottle, and add water 4.5ml, ultrasonic process 30 minutes, take out, let cool, add methyl alcohol to scale, shake up, use 0.45um filtering with microporous membrane, get the analysis of subsequent filtrate sample introduction;

B) ginkalide A reference substance is got in the preparation of reference substance solution, ginkolide B reference substance, ginkalide C reference substance, Bilobalide reference substance are appropriate, add the mixed solution that 50% methyl alcohol makes each bilobalide-containing A0.18mg of every 1ml, ginkolide B 0.08mg, ginkalide C 0.1mg, Bilobalide 0.2mg respectively, to obtain final product;

Chromatographic condition and system flexibility octadecylsilane chemically bonded silica are filling agent; Volume ratio is the methyl alcohol-tetrahydrofuran-water of 25:10:65 is mobile phase, and evaporation scatter detector detects.Number of theoretical plate calculates by Bilobalide peak and is not less than 3000.

3. quality determining method according to claim 1 and 2, is characterized in that in content assaying method, and compound silver Lingtong capsule composition containing ginkgo leaf is with the total amount of ginkalide A, ginkolide B, ginkalide C and Bilobalide, and every must not be less than 0.44mg.

4. quality determining method according to claim 1, is characterized in that in described discrimination method, and comprise glossy ganoderma discriminating, radix scrophulariae is differentiated, grass-leaved sweetflag is differentiated, concrete steps are as follows:

5. quality determining method according to claim 1, is characterized in that in described discrimination method, and comprise glossy ganoderma discriminating, radix scrophulariae is differentiated, grass-leaved sweetflag is differentiated, concrete steps are as follows:

6. quality determining method according to claim 4, is characterized in that glossy ganoderma discrimination method specifically comprises the following steps:

Get compound silver Lingtong capsule content 3g, add chloroform backflow twice, each 50ml, first time backflow 3 hours, second time backflow 1 hour, merging filtrate, filtrate evaporate to dryness, residue adds methyl alcohol 1ml makes dissolving, as need testing solution.Separately get glossy ganoderma control medicinal material 0.5g, be made in the same way of control medicinal material solution.According to thin-layered chromatography, drawing each 4 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, is that the toluene-ethyl acetate-acetic acid mixed solution of 12:12:0.5 is developping agent with volume ratio, launch, take out, dry, spray with 10% ethanol solution of sulfuric acid, spot development is heated to clear at 105 DEG C, in test sample chromatogram, on the position corresponding to control medicinal material chromatogram, the spot of aobvious same color.

7. quality determining method according to claim 4, is characterized in that radix scrophulariae discrimination method specifically comprises the following steps:

Get compound silver Lingtong capsule content 6g, add methyl alcohol 25ml, soak 1 hour, ultrasonic process 30 minutes, filter, filtrate evaporate to dryness, the residue 25ml that adds water makes dissolving, with water saturated normal butyl alcohol shaking out 2 times, each 30ml, merges normal butyl alcohol liquid, evaporate to dryness, residue adds methyl alcohol 5ml makes dissolving, as need testing solution; Separately get radix scrophulariae control medicinal material 2g, be made in the same way of control medicinal material solution.Get harpagoside reference substance again, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.According to thin-layered chromatography, drawing each 4 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, is that lower floor's solution of the chloroform-methanol-water mixed solution of 12:4:1 is developping agent with volume ratio, launch, take out, dry, spray with 5% vanillin-sulfuric acid ethanolic solution, spot development is heated to clear at 105 DEG C, in test sample chromatogram, on the position corresponding with control medicinal material chromatogram to reference substance chromatogram, the spot of aobvious same color.

8. quality determining method according to claim 4, is characterized in that grass-leaved sweetflag discrimination method specifically comprises the following steps: