CN105004833B - It is a kind of treat acute gouty arthritis, gout Chinese medicine preparation detection method - Google Patents
It is a kind of treat acute gouty arthritis, gout Chinese medicine preparation detection method Download PDFInfo
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Abstract
The invention discloses it is a kind of treat acute gouty arthritis, gout Chinese medicine preparation detection method, which is made any dosage form of pharmacy by 1~10 part of 1~30 part of desmodium, 1~20 part of the root of Chinese clematis, 1~20 part of Piper wallichii (Miq.) Hand.-Mazz., 1~20 part of starflowerlike loosestrife root or herb, 1~20 part of siphonostegia chinensis, 1~20 part of rhizoma alismatis and Asiatic plantain or more 7 taste Chinese medicines.There is heat-clearing dissipating bind, inducing diuresis for treating strangurtia, blood-activating analgetic, for the numbness disease symptoms include extremities joint pain caused by cold-dampness impatency channels and collaterals, waist and knee crymodynia, joint stuffiness；Acute gouty arthritis and gout interictal are shown in above-mentioned patient.Detection method provided by the invention differentiates desmodium, the root of Chinese clematis, rhizoma alismatis in preparation for thin layer；Kaempferol in assay desmodium.The quality determining method can effectively control the true and false of product and the quality of quality, ensure that safety, the validity of patient medication.
Description
Technical field
The present invention relates to it is a kind of treat acute gouty arthritis, the Chinese medicine preparation of gout (releive glue by trade name gout
Capsule) detection method.
Background technology
Gout due to guanine metabolic disorder and underexcretion, makes uric acid content continue to increase and cause lithate
Joint part is deposited on, is inflamed reaction in extraneous irritation, damaging tissue and the clinical syndrome that generates.Its clinical symptoms
Mainly joint part is red, hot, swollen, pain, and the gold dollar age traditional Chinese medical science just has understanding to " gout ", and emphasizing the cause of disease of gout is
It is attacked outside wind-cold damp pathogen gas, and to understanding of the gout modern Chinese medicine based on forefathers, there are many different explanation reasons, summary
Reason, the author think the more organism inadequate natural endowments of patient with gout, and the weary void of spleen kidney, the day after tomorrow, undesirable diet, ate delicious food thickness, so that wet
Hot inner product, easily by being attacked outside ailment said due to cold or exposure, inside and outside to accompany each other, damp and hot ailment said due to cold or exposure is stranded in joint, and easy blood vessels are obstructed, finally develop into goat.
The initial clinical symptoms of gout are acute gouty arthritis, and performance is the acute single joint or more passes to break out at night
Save pain.It is the cause of disease of acute gouty arthritis into the long-pending Monosodium urate crystallization in joint part.Due to some, as bird is fast
Purine metabolic disorder and underexcretion etc., the content for making uric acid in blood is higher, in some joints and periarticular tissue, by
Few, the easy anoxic in the position blood vessel easily increases the generation of lactic acid and reduces the pH of joint part, and the matrix of joint part
In containing more connective tissue and glutinous polysaccharide acid, therefore lithate be easier to joint part deposit.Uric acid sodium salt crystallizes into product
After joint part, under the influence of external condition, such as it is cold, drink, stimulate mast cell degranulation and macrophage activation
Increasing, discharge cell factor and chemotactic factor (CF), activation neutrophil leucocyte release Superoxide dismutase discharges more adhesion molecules,
A large amount of cell factor and chemotactic factor (CF), etc. is discharged to cause nonspecific inflammatory reaction and damage joint part.Blood uric acid
Value is unexpected to be raised and lowered, and can all cause inflammatory reaction.Because rapidly raising makes uric acid crystal be deposited on pass to blood uric acid value
Save position；And quickly reduce, make to be deposited on the dissolving of joint part lithate.
Traditional Chinese medical science field has gout treatment unique one side, my company discloses publication number on 06 22nd, 2010
The patent of CN101926896A provides a kind of Chinese medicine preparation for treating gout and preparation method thereof, and the Chinese medicine preparation is by money
10 parts of 30 parts of grass, 15 parts of the root of Chinese clematis, 10 parts of Piper wallichii (Miq.) Hand.-Mazz., 15 parts of starflowerlike loosestrife root or herb, 15 parts of siphonostegia chinensis, 12 parts of rhizoma alismatis and Asiatic plantain are made
Any dosage form in pharmacy, there is apparent anti-inflammatory effect and analgesic activity, between acute gouty arthritis and gout breaking-out
The various illnesss of phase have good therapeutic effect, and uneasy to recur, no obvious toxic-side effects and adverse reaction.
The present invention is existing to provide a kind of detection method to the Chinese medicine preparation, and the detection method is reliable and stable, can effectively ensure to produce
Quality.
Invention content
To solve the deficiencies in the prior art, acute gouty arthritis, pain are treated the purpose of the present invention is to provide a kind of
The detection method of the Chinese medicine preparation of wind, detection method is simple and convenient and reliable and stable, can be effectively detected desmodium,
A variety of active principles such as the root of Chinese clematis, rhizoma alismatis, effectively ensure product quality.
In order to realize above-mentioned target, the present invention adopts the following technical scheme that：It is a kind of to treat acute gouty arthritis, pain
The detection method of the Chinese medicine preparation of wind, the detection method includes differentiating and/or the measure project of content；Wherein differentiate it is to gold
Money grass, the root of Chinese clematis and/or the indentification by TLC of rhizoma alismatis；Assay is with desmodium in Preparations by HPLC
Content.
Above-mentioned treatment acute gouty arthritis, gout Chinese medicine preparation detection method in, the discriminating side of desmodium
Method is specially：The Chinese medicine preparation of 3~4g crude drug powders is taken, adds 80% 30~50ml of methanol, is heated to reflux 0.5~1 hour, puts
Cold, filtration, filtrate is evaporated, and residue adds 8~10ml of water to make dissolving, is extracted 2~4 times with the ether medicine that shakes, 10~30ml, discards every time
Ether solution, aqueous add 8~10ml of dilute hydrochloric acid, put in water-bath and heat 0.5~1 hour, take out, rapid to cool down, and are shaken with ethyl acetate
Extraction 2~4 times is shaken, every time 20~30ml, combined ethyl acetate is washed with 30~50ml of water, discards aqueous, and acetic acid ethyl fluid steams
Dry, residue adds 1~2ml of methanol to make dissolving, as test solution；Desmodium 0.5~1.5g of control medicinal material separately is taken, is made in the same way of
Control medicinal material solution；It is tested according to thin-layered chromatography (VI B of annex), draws 3~4 μ l of test solution, 3~4 μ l of reference substance solution,
It is put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid=10~8：8~6：1~0.5 is solvent, is opened up
It opens, takes out, dry, spray with 5% alchlor ethanol solution, heated several minutes at 105 DEG C, put and examined under the ultraviolet lamp of 365nm
Depending on；In test sample chromatography, on control medicinal material corresponding position, showing the fluorescence spot of same color.
Aforementioned treatment acute gouty arthritis, gout Chinese medicine preparation detection method in, the discriminating side of the root of Chinese clematis
Method is specially：The medicine Chinese medicine preparation of 2~3g crude drug powders is taken, adds 30~50ml of ethyl alcohol, is heated to reflux 1~2 hour, is filtered, filter
Liquid is concentrated into 15~20ml, adds 3~6ml of hydrochloric acid, is heated to reflux 0.5~1 hour, adds 8~10ml of water, let cool, and adds in 60~90
DEG C 15~25ml of petroleum ether shaking extraction 3~4 times, every time 20~25ml, petroleum ether be evaporated, residue 7~10ml of absolute ethyl alcohol
Dissolving, as test solution；Oleanolic acid reference substance separately is taken, adds absolute ethyl alcohol that solution of the 1ml containing 0.45mg is made, as right
According to product solution；It is tested according to thin-layered chromatography (VI B of annex), draws 7~8 μ l of test solution, 2~3 μ l of reference substance solution, respectively
Point is on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid=20~17：3~1.5：0.2~0.1 is solvent, thin
Laminate puts presaturation 5~10 minutes in expansion cylinder, is unfolded, and takes out, dries, spray with 10% ethanol solution of sulfuric acid, in 105 DEG C of heating
It is clear to spot development；In test sample chromatography, on position corresponding with reference substance chromatography, the spot of same color is shown.
Aforementioned treatment acute gouty arthritis, gout Chinese medicine preparation detection method in, the discrimination method of rhizoma alismatis
Specially：The medicine Chinese medicine preparation of 8~10g crude drug powders is taken, adds 40~50ml of ethyl alcohol, stirs evenly, is ultrasonically treated 70~90 minutes, filter
It crosses, filtrate is evaporated, and residue adds 40~50ml of water to make dissolving, filters off not tolerant, and filtrate extracts 3 with 60~90 DEG C of petroleum ether shaking
~4 times, 40~50ml, merges petroleum ether liquid, is evaporated every time, and residue adds 60~90 DEG C of petroleum ether 1ml to make dissolving, as trying
Product solution；Rhizoma alismatis 1.5~2g of control medicinal material separately is taken, adds 40~50ml of water, is heated to reflux 30 minutes, lets cool, filtered with absorbent cotton,
Filtrate shakes extraction 3~4 times with petroleum ether, and 30~50ml (centrifuging when necessary), merges petroleum ether extract, be evaporated every time, residual
Slag adds 60~90 DEG C of 0.5~1ml of petroleum ether to make dissolving, as control medicinal material solution；It is tried according to thin-layered chromatography (VI B of annex)
It tests, draws each 9~12 μ l of above two solution, put respectively on same silica gel g thin-layer plate, with 60~90 DEG C of petroleum ether-three
Chloromethanes-ethyl acetate=2~1：1~0.5：2~1 be solvent, is unfolded, takes out, dry, spray molten with 5% phosphomolybdic acid ethyl alcohol
It is clear to be heated to spot development at 110 DEG C for liquid；In test sample chromatography, on control medicinal material corresponding position, showing same color
Spot.
Aforementioned treatment acute gouty arthritis, gout Chinese medicine preparation detection method in, desmodium assay
Method is using Kaempferol reference substance as control, with -0.4% phosphoric acid solution=50 of methanol：50 be mobile phase be the efficient of mobile phase
Liquid chromatography.
Aforementioned treatment acute gouty arthritis, gout Chinese medicine preparation detection method in, desmodium containing measuring
The method of determining is specially：
The assay of Kaempferol in desmodium：
Chromatographic condition is with system suitability using octadecylsilane chemically bonded silica as filler；With -0.4% phosphorus of methanol
Acid solution=50：50 be mobile phase；Detection wavelength is 360nm；Plate number is calculated by Kaempferol not less than 2500；
The preparation of reference substance solution；Take Kaempferol reference substance appropriate, it is accurately weighed, add 80% methanol that every 1ml is made containing mountain
How the solution of 20 μ g of element to get；
The preparation of test solution；The medicine Chinese medicine preparation for being equivalent to 1.5g crude drug powders is taken, it is accurately weighed, put tool plug taper
In bottle, precision adds in 80% methanol 50ml, and close plug, weighed weight is heated to reflux 1 hour, lets cool, then weighed weight, with 80%
Methanol supplies the weight of less loss, shakes up, filtration；Precision measures subsequent filtrate 25ml, and precision adds in hydrochloric acid 5ml, puts in 90 DEG C of water-baths
Heating hydrolysis 1 hour, takes out, rapid to cool down, and is transferred in 50ml measuring bottles, with 80% methanol dilution to scale, shakes up, and filters,
Take subsequent filtrate to get；
Measuring method；It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, liquid chromatograph is injected, is measured,
To obtain the final product.
Aforementioned treatment acute gouty arthritis, gout Chinese medicine preparation detection method in, the Chinese medicine preparation is by weight
Part is measured by 1~30 part of desmodium, 1~20 part of the root of Chinese clematis, 1~20 part of Piper wallichii (Miq.) Hand.-Mazz., 1~20 part of starflowerlike loosestrife root or herb, siphonostegia chinensis 1~20
1~10 part of part, 1~20 part of rhizoma alismatis and Asiatic plantain are made of bulk pharmaceutical chemicals.
Aforementioned treatment acute gouty arthritis, gout Chinese medicine preparation detection method in, the Chinese medicine preparation is by weight
Part is measured by 30 parts of desmodium, 15 parts of the root of Chinese clematis, 10 parts of Piper wallichii (Miq.) Hand.-Mazz., 15 parts of starflowerlike loosestrife root or herb, 15 parts of siphonostegia chinensis, 12 parts of rhizoma alismatis and Chinese herbaceous peony
10 parts of grass is made.
Aforementioned treatment acute gouty arthritis, gout Chinese medicine preparation detection method in, take desmodium with 75%
Alcohol reflux extracts, filtration, and recycling ethyl alcohol is simultaneously concentrated into appropriate, obtains the dregs of a decoction and alcoholic solution, by the dregs of a decoction and the root of Chinese clematis, Piper wallichii (Miq.) Hand.-Mazz.,
Starflowerlike loosestrife root or herb, siphonostegia chinensis, rhizoma alismatis and Asiatic plantain add water to cook three times, and filtration, filtrate merges with alcoholic solution, is concentrated under reduced pressure into thick
Cream dry, pulverize, sieving, mix with auxiliary material, be sieved, mixing, loading capsule, be made 1000 to get.
Aforementioned treatment acute gouty arthritis, gout Chinese medicine preparation detection method in, the Chinese medicine preparation
Any conventional formulation can be prepared into, including tablet, pill, capsule, electuary, oral liquid, spray, injection, suspension
Liquid, microball preparation
In order to ensure detection method science, reasonable, feasible, the detection method of the present invention is studied and examined
It examines.
First, the content assaying method of desmodium is investigated：
Desmodium is releived the monarch drug in a prescription in capsule (invention formulation) for gout, and Kaempferol is its principle active component, therefore is selected
The index components of the inherent quality using Kaempferol as this preparation are selected, pre-treatment sample are carried out using sour water solution, using efficient liquid phase
Chromatography carries out sample measure, through experiment, the results showed that, this method is easy to operate, at low cost, and Check-Out Time is short, has simultaneously
Good separating effect, it is sensitive, accurate the advantages that.
1st, instrument, reagent and sample
Agilent Technologies 1200series high performance liquid chromatographs, KQ-250DB type numerical control supersonics are clear
Wash device (Kunshan Ultrasonic Instruments Co., Ltd.), ten a ten thousandth electronic balances (plum Teller-support benefit instrument (Shanghai) limited public affairs
Department), methanol (analysis pure 20130411, Shanghai Shen Bo Chemical Co., Ltd.s), (analysis pure 20140328, chemical industry is won in Shanghai Shen to HCl
Co., Ltd), acetonitrile (chromatographically pure 20140721, Shanghai Shen Bo Chemical Co., Ltd.s), phosphoric acid (analysis pure 20140421, Shanghai
Shen Bo Chemical Co., Ltd.s), (Nat'l Pharmaceutical ＆ Biological Products Control Institute, for assay, number is Kaempferol reference substance
112542-201405), other are that analysis is pure.Gout releive capsule (20140304,20140201,20140202,
20140401st, 20140402,20140403,20140404,20140405,20140501,20140502,20140503, this hair
Bright preparation is provided by Guizhou lark Pharmacy stock Co., Ltd).
2nd, chromatographic condition and system suitability test
Using octadecylsilane chemically bonded silica as filler；With -0.4% phosphoric acid solution (50 of methanol：50) it is mobile phase；Inspection
Survey wavelength is 360nm.Number of theoretical plate is calculated by Kaempferol not less than 2500.
3rd, the preparation of reference substance solution
Take Kaempferol reference substance appropriate, it is accurately weighed, add 80% methanol that the solution of every 1ml 20 μ g containing Kaempferol is made, i.e.,
.
4th, the preparation of test solution
This product content about 1.5g is taken, it is accurately weighed, it puts in conical flask with cover, precision adds in 80% methanol 50ml, close plug,
Weighed weight is heated to reflux 1 hour, lets cool, then weighed weight, and the weight of less loss is supplied with 80% methanol, is shaken up, filtration.Essence
Close measurement subsequent filtrate 25ml, precision add in hydrochloric acid 5ml, put heating hydrolysis 1 hour in 90 DEG C of water-baths, take out, rapid to cool down, transfer
Into 50ml measuring bottles, with 80% methanol dilution to scale, shake up, filter, take subsequent filtrate to get.
5th, the preparation of negative control solution
In prescription ratio and preparation process, the negative sample without desmodium medicinal material is prepared, feminine gender is prepared with method by " 5 "
Sample solution.
6th, methodology validation
6.1st, system suitability test
According to above-mentioned chromatographic condition and sample treatment, system suitability test, Kaempferol peak and other compositions are carried out
Reach baseline separation, and peak shape is good；Negative sample result of the test shows that other ingredients are noiseless to the measure of Kaempferol.As a result
See Fig. 1, Fig. 2, Fig. 3.
6.2nd, the investigation of linearity and range
5.0,10.0,15.0,20.0, the 25.0 μ L of reference substance solution under 4 is taken to distinguish sample introduction, are remembered by above-mentioned chromatographic condition
Chromatogram is recorded, measures peak area, using the logarithm of peak area as ordinate, the logarithm of Kaempferol reference substance sample size is horizontal seat
Mark carries out linear regression, obtains regression equation：Y=1.1465X+4.5823 (Y is peak area, and X is concentration), r=0.9999.Knot
Fruit shows that Kaempferol is in good linear relationship with integrating peak areas value in 5.5850~27.9250 μ g ranges.
6.3rd, precision test
The reference substance solution under 4 is taken, is tested by 3 chromatographic conditions, sample size is 20 μ l, continuous sample introduction 5 times,
Its integrating peak areas value is measured, RSD 0.72% shows that instrument precision is good.
6.4th, repetitive test
The gout of same lot number is taken to releive capsule (lot number：20140304), parallel 6 parts of sampling is operated by 3 lower methods
And it detects.Kaempferol average mass fraction is 5.37mg/g, RSD 0.10%, shows method repeatability preferably.
6.5th, stability test
Take the test solution (lot number of same lot number：20140304), tested by 3 chromatographic conditions, in 0,4,8,
12nd, 16,20, each sample introduction is primary for 24 hours, and sample size is 20 μ L, is calculated by integrating peak areas value, and the RSD values of Kaempferol are
0.14%, show that Kaempferol is interior for 24 hours with good stability in test solution.
6.6th, sample recovery rate is tested
9 parts of the sample (mass fraction 5.28mg/g) of known kaempferia galamga cellulose content is weighed, is divided into 3 groups (every group 3 parts), puts cone
In shape bottle, the accurate Kaempferol reference substance solution 4 for adding in 2.5204mg/mL of every group of difference, 5,6mL are accurately weighed, by under 5
It from " precision adds in 80% methanol 50mL ", is operated with method, and by 3 lower chromatographic condition sample introductions, sample size is 20 μ l, is measured
Peak area calculates the rate of recovery.It the results are shown in Table 1.
1 Kaempferol sample recovery rate result of the test (n=9) of table
By 1 interpretation of result of table, sample recovery rate mean value is 99.90%, RSD 1.57%, shows the accuracy of this method
It is high.
6.7th, serviceability test
It chooses in experiment to 3 different mobile phase ratios, column temperature and flow velocity factors, according to processing side under " 4 " and " 5 " item
Method respectively investigates above-mentioned factor.
6.7.1, the investigation of different mobile phase ratios
Chromatographic column：Venusil C18 columns (4.6mm × 250mm, 5 μm)；Volume flow 0.8mL/min；10 μ l of sample size.
Sample introduction is distinguished according to reference substance solution under " 4 " and " 5 " item and with a collection of (20140304) test solution, by the not cocurrent flow of table 2
Dynamic phase composition ratio is measured, and the results are shown in Table 2.
The different mobile phase composition ratio of table 2 are investigated
By table 2, the result shows that, mobile phase is -0.4% phosphoric acid=60~50 of methanol:40~50 float up and down, and RSD values are
0.38%, illustrate the content of Kaempferol in still Accurate Determining sample.
6.7.2, the investigation of different column temperatures
Chromatographic column：Venusil C18 columns (4.6mm × 250mm, 5 μm)；- 0.4% phosphoric acid solution (50 of methanol：50) it is stream
Dynamic phase；Volume flow 0.8mL/min；10 μ l of sample size.According to reference substance solution and a collection of together under " 4 " and " 5 " item
(20140304) test solution difference sample introduction is measured by the different column temperatures of table 3, the results are shown in Table 3.
The different column temperatures of table 3 are investigated
By table 3, the result shows that, column temperature floats up and down at 25 DEG C~30 DEG C, and RSD values are 0.11%, illustrate the short and small change of column temperature
Change, still in Accurate Determining sample Kaempferol content.
6.7.3, investigation different in flow rate
Chromatographic column：Venusil C18 columns (4.6mm × 250mm, 5 μm)；- 0.4% phosphoric acid solution (50 of methanol：50) it is stream
Dynamic phase；10 μ l of sample size.Distinguish according to reference substance solution under " 4 " and " 5 " item and with a collection of (20140304) test solution
Sample introduction is measured by the different column temperatures of table 4, the results are shown in Table 4.
4 investigation different in flow rate of table
By table 4, the result shows that, for flow velocity in the range of 1.0~0.4ml/min, RSD values are 0.11%；In 1.5ml/min waves
It is dynamic larger.Therefore flow velocity is chosen in 1.0~0.4ml/min ranges, appropriate, the content of Kaempferol in energy Accurate Determining sample.
6.8th, 11 batches of sample assay results
11 batches of samples are taken, sample are handled by 5 lower methods, as test solution.With 0.45 μm of filtering with microporous membrane, respectively
10 μ L of subsequent filtrate are taken, inject high performance liquid chromatograph, are measured by 3 chromatographic conditions, sample is calculated with external standard two-point method logarithmic equation
The content of Kaempferol, the results are shown in Table 5 in product.
5 11 batches of gouts of table are releived capsule Kaempferol assay result (n=3)
By 5 interpretation of result of table, minimum event 5.19mgg containing Kaempferol is chosen- 130% is floated downward, content limit can be determined
For：In terms of Kaempferol, 3.5mg must not be less than per 1g containing desmodium for capsule for treating prostatitis.
2nd, the discriminating of desmodium：
Desmodium is the drying herb of Ofthe Primulaceae desmodium Lysimachia christinae Hance, is recorded
In version Chinese Pharmacopoeia (one) in 2010.Desmodium is releived the monarch drug in a prescription of prescription as gout, this experiment will choose desmodium control
Medicinal material is first cleaned using extraction as control, after releived desmodium in capsule with two-phase acid-hydrolyzed method extraction gout
Characteristic chemical constituent carries out thin layer discriminating to desmodium medicinal material, and it is thin to investigate different expansion media, different point sample amounts, different manufacturers
The influence of laminate, different temperatures, different humidity to desmodium medicinal material thin-layer chromatography in said preparation.Result of the test shows：With money
Careless control medicinal material is reference substance, and toluene-ethyl acetate-formic acid (10: 8: 1) is development system, and desmodium medicinal material thin layer is differentiated
Feature is apparent, and specificity is strong, the thin-layer identification method of desmodium medicinal material in the capsule that can releive as gout, therefore is included in inspection of the present invention
Survey project.
2.1 instruments, reagent and sample
KQ-250DB types numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), DGG-9246A electric heating constant temperatures
Air dry oven (Shanghai Qi Xin scientific instrument Co., Ltd), ten a ten thousandth electronic balances (plum Teller-support benefit instrument (on
Sea) Co., Ltd), methanol (analysis pure 20130411, Shanghai Shen Bo Chemical Co., Ltd.s).(analysis is pure for ethyl acetate
20140528, Shanghai Shen Bo Chemical Co., Ltd.s), concentrated hydrochloric acid (analysis pure 20130721, Shanghai Shen Bo Chemical Co., Ltd.s), gold
Money grass control medicinal material (Nat'l Pharmaceutical ＆ Biological Products Control Institute, for differentiating, number 111532-201402), gout is releived glue
(20140304,20140201,20140202, invention formulation is carried capsule by Braun Guizhou Pharmaceutical Enterprise Group Co
For).
It is prepared by 2.2 test solutions
This product content 3g is taken, adds 80% methanol 50ml, is heated to reflux 1 hour, lets cool, is filtered, filtrate is evaporated, and residue adds
Water 10ml makes dissolving, with ether shake medicine extract 2 times, each 10ml, discard ether solution, aqueous adds dilute hydrochloric acid 10ml, puts in water-bath
Heating 1 hour is taken out, rapid to cool down, with ethyl acetate shaking extraction 2 times, each 20ml, combined ethyl acetate, with water 30ml
Washing, discards aqueous, acetic acid ethyl fluid is evaporated, and residue adds methanol 2ml to make dissolving.
The preparation of 2.3 reference substance solutions
Desmodium control medicinal material 0.5g is taken, by " 2.2 " same treatment.
The preparation of 2.4 negative samples
In prescription ratio and preparation process, the negative sample without desmodium medicinal material is prepared, the moon is prepared with method by " 2.2 "
Property sample solution.
2.5 development system
Toluene-ethyl acetate-formic acid (10：8：1).
2.6 color developing agent
5% alchlor ethanol solution.
The investigation of 2.7 thin-layer chromatography conditions
2.7.1 the selection of lamellae
4 μ l of 3 μ l of test solution and reference substance solution are drawn respectively, with 2 kinds of differences of thin layer silica G plate and polyamide film
Types of material lamellae, is unfolded, and is taken out, is dried, spray with color developing agent, and 105 DEG C of heating 5min are examined under 365nm ultraviolet lamps
Depending on.See Fig. 4, Fig. 5.
By Fig. 4, Fig. 5 as it can be seen that polyamide film plate does not detach test sample；Silica G plate good separation, spot circle
It is whole, on control medicinal material chromatography corresponding position, showing same color spot.Therefore choose the expansion of silica G plate.
2.7.2 best point sample amount is investigated
1,2,3,4,5 μ l of test solution and reference substance solution are drawn respectively, are put respectively on different thin layer silica G plates,
It is investigated, is unfolded, taken out, dry, spray with color developing agent, 105 DEG C of heating 5min are inspected under 365nm ultraviolet lamps.See Fig. 6, figure
7。
By Fig. 6, Fig. 7 as it can be seen that 3 μ l point sample amount best results of test sample, when point sample amount is more than 3 μ l, test sample principal spot
There is trailing phenomenon；Best 4~6 μ l of point sample amount of reference substance all can, therefore select point sample amount as 4 μ l.
Experimental result is investigated with reference to above-mentioned 2 kinds of factors, it may be determined that lamellae is silica G plate, test sample and best point sample amount
For 3 μ l, the best point sample amount of reference substance is 4 μ l.It is verified through preliminary experiment, determines the thin-layer identification method of desmodium, and according to《China
Pharmacopeia》2010 editions Ⅹ VIII A quality standards in Chinese drugs analysis method verification guides principles of an annex carry out methodology validation.
2.8 methodology validation
2.8.1 specificity is investigated
It is releived capsule sample with the gout of three batches, carries out specificity experiment according to the above method, as a result see Fig. 8.
As seen from Figure 8, negative sample is noiseless, and principal spot Rf values are about 0.42；With control medicinal material chromatography corresponding position
On, show the clear spot of same color, spot rounding, good separating effect.Show that this method specificity is strong, spot development is clear.
2.8.2 serviceability test
It is releived capsule sample, 3 different manufacturers lamellaes is compared respectively, 3 kinds of differences with the gout of three batches
Temperature and humidity are tested as variable according to above-mentioned discrimination method.
2.8.2.1 different manufacturers lamellae is tested, and as a result sees Fig. 9-Figure 11.
3 different manufacturers durabilities the experimental results showed that, gout is releived the thin layer discriminating figure principal spot of desmodium in capsule
Separating effect does very well, spot rounding, and different manufacturers lamellae influences principal spot less, and repeatability is preferably.
2.8.2.2 different temperatures and humid test
Different temperatures and humidity set parameter are shown in Table 1.Experiment collection of illustrative plates is shown in Figure 12-Figure 14.
The investigation of 1 different temperatures of table and relative humidity
By the investigation experimental patterns of different temperatures and relative humidity as it can be seen that in test sample chromatography, with reference substance chromatography
On corresponding position, the fluorescence spot of same color is shown, clear spot, good separating effect, Rf values are moderate, and negative control is without dry
It disturbs.It follows that temperature and relative humidity change in usual environmental field, the validity of the discrimination method is not influenced.
3rd, the discriminating of the root of Chinese clematis
The root of Chinese clematis is ranunculaceae plant root of Chinese clematis Clematis chinensis Osbeck, Clematis hexapetala
The dry root and rhizome of Clematishexa petala Pall. or northeast clematis Clematis manshurica Rupr..
It records in version Chinese Pharmacopoeia (one) in 2010.The root of Chinese clematis is releived the ministerial drug of prescription as gout, and oleanolic acid is main for its
Effective active composition, therefore, this experiment will choose oleanolic acid as index ingredient with reference to Chinese Pharmacopoeia version one in 2010
TLC methods under " root of Chinese clematis " are tested, and thin layer discriminating is carried out, and investigate difference using oleanolic acid reference substance as control
Sample amount, different manufacturers lamellae, different temperatures and different humidity releive to gout the shadow of clematis chinensis medicinal materials thin-layer chromatography in capsule
It rings.Result of the test shows using oleanolic acid reference substance as control, toluene-ethyl acetate-formic acid (20:3:0.2) it is expansion system
System, apparent to clematis chinensis medicinal materials thin layer diagnostic characteristics, specificity is strong.Therefore in the capsule that can releive as gout clematis chinensis medicinal materials it is thin
Layer discrimination method, therefore it is included in detection project of the present invention.
3.1 instruments, reagent and sample
KQ-250DB types numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), DGG-9246A electric heating constant temperatures
Air dry oven (Shanghai Qi Xin scientific instrument Co., Ltd), ten a ten thousandth electronic balances (plum Teller-support benefit instrument (on
Sea) Co., Ltd), ethyl alcohol (analysis pure 20130411, Shanghai Shen Bo Chemical Co., Ltd.s), petroleum ether (analysis pure 20140318,
Shanghai Shen Bo Chemical Co., Ltd.s), toluene (analysis pure 20140721, Shanghai Shen Bo Chemical Co., Ltd.s), oleanolic acid control
(Nat'l Pharmaceutical ＆ Biological Products Control Institute, for assay, number 112542-201405), gout are releived capsule
(20140304,20140201,20140202, invention formulation is carried by Braun Guizhou Pharmaceutical Enterprise Group Co
For).
It is prepared by 3.2 test solutions
This product content 2g is taken, adds ethyl alcohol 50ml, is heated to reflux 2 hours, is filtered, filtrate is concentrated into 20ml, adds hydrochloric acid
3ml is heated to reflux 1 hour, adds water 10ml, let cool, and adds petroleum ether (60~90 DEG C) 25ml shakings extraction 4 times, each 25ml, stone
Oily ether is evaporated, and residue absolute ethyl alcohol 10ml dissolves.
The preparation of 3.3 reference substance solutions
Oleanolic acid reference substance is taken, adds absolute ethyl alcohol that solution of the 1ml containing 0.45mg is made.
The preparation of 3.4 negative samples
In prescription ratio and preparation process, the negative sample without clematis chinensis medicinal materials is prepared, the moon is prepared with method by " 3.2 "
Property sample solution.
3.5 development system
Toluene-ethyl acetate-formic acid (20：3：0.2).
3.6 color developing agent
10% ethanol solution of sulfuric acid.
3.7 best point sample amounts are investigated
1,2,3,5,7,10,11 μ l of test solution, 1,2,3,4,5,6,7 μ l of reference substance solution are drawn respectively, respectively point
It on different thin layer silica G plates, is investigated, is unfolded, taken out, dry, spray with color developing agent, 105 DEG C of heating 5min, in 365nm
It is inspected under ultraviolet lamp.See Figure 15, Figure 16.
By Figure 15, Figure 16 as it can be seen that the best 7 μ l of point sample amount of test sample, the best point sample amount of control medicinal material are 3 μ l.
3.8 methodology validation
3.8.1 specificity is investigated
It is releived capsule sample with the gout of three batches, specificity experiment, the result is shown in Figure 17 is carried out according to the above method.
As seen from Figure 17, negative sample is noiseless；With on reference substance chromatography corresponding position, showing the clear spot of same color
Point, spot rounding, good separating effect.Show that this method specificity is strong, spot development is clear.
3.8.2 serviceability test
It is releived capsule sample, 3 different manufacturers lamellaes is compared respectively, 3 kinds of differences with the gout of three batches
Temperature and humidity are tested as variable according to above-mentioned discrimination method.
3.8.2.1 different manufacturers lamellae is tested, the result is shown in Figure 1 8- Figure 20.
3 different manufacturers durabilities the experimental results showed that, the gout thin layer of the root of Chinese clematis in capsule of releiving differentiates figure spot point minute
Do very well from effect, spot rounding, different manufacturers lamellae influences root of Chinese clematis principal spot less, and repeatability is preferably.
3.8.2.2 different temperatures and humid test
Different temperatures and humidity set parameter are shown in Table 1.Experiment collection of illustrative plates is shown in Figure 21-Figure 23.
The investigation of 1 different temperatures of table and relative humidity
By the investigation experimental patterns of different temperatures and relative humidity as it can be seen that in test sample chromatography, with reference substance chromatography
On corresponding position, the fluorescence spot of same color is shown, clear spot, good separating effect, Rf values are moderate, and negative control is without dry
It disturbs.It follows that temperature and relative humidity change in usual environmental field, the validity of the discrimination method is not influenced.
4th, the discriminating of rhizoma alismatis
Rhizoma alismatis is labiate hair leaf lycopus lucidus Lycopus lucidus Turcz.Var.hirtus Regel's
Dry aerial parts are recorded in version Chinese Pharmacopoeia (one) in 2010.Rhizoma alismatis is releived the medicine that makes of prescription as gout, this experiment will
Rhizoma alismatis control medicinal material is chosen as control, first using the abundant ultrasonic extraction characteristic chemical constituent of ethyl alcohol, the rear method with extraction is extracted
Gout is releived the characteristic chemical constituent of rhizoma alismatis in capsule, carries out thin layer discriminating to Rhizoma Alismatis, and investigate different point sample amounts, different factories
The influence of family lamellae, different temperatures, different humidity to Rhizoma Alismatis thin-layer chromatography in said preparation.Result of the test shows：With pool
Control medicinal material is rushed down for reference substance, petroleum ether (60~90 DEG C)-chloroform-ethyl acetate (2：1：2) it is development system, to rhizoma alismatis
Medicinal material thin layer diagnostic characteristics are apparent, and specificity is strong, the thin-layer identification method of Rhizoma Alismatis in the capsule that can releive as gout, therefore arrange
Enter detection project of the present invention.
4.1 instruments, reagent and sample
KQ-250DB types numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), DGG-9246A electric heating constant temperatures
Air dry oven (Shanghai Qi Xin scientific instrument Co., Ltd), ten a ten thousandth electronic balances (plum Teller-support benefit instrument (on
Sea) Co., Ltd), petroleum ether (analysis pure 20130411, Shanghai Shen Bo Chemical Co., Ltd.s).Ethyl alcohol (analysis pure 20140528,
Shanghai Shen Bo Chemical Co., Ltd.s), chloroform (analysis pure 20130721, Shanghai Shen Bo Chemical Co., Ltd.s), rhizoma alismatis control
Medicinal material (Nat'l Pharmaceutical ＆ Biological Products Control Institute, for differentiating, number 111532-201403), gout is releived capsule
(20140304,20140201,20140202, invention formulation is carried by Braun Guizhou Pharmaceutical Enterprise Group Co
For).
It is prepared by 4.2 test solutions
This product content 10g is taken, adds ethyl alcohol 50ml, stirs evenly, is ultrasonically treated 90 minutes, filtration, filtrate is evaporated, and residue adds water
50ml makes dissolving, filters off not tolerant, and filtrate (60~90 DEG C) shaking extractions 4 times, each 50ml of petroleum ether merge petroleum ether
Liquid is evaporated, and residue adds petroleum ether (60~90 DEG C) 1ml to make dissolving.
The preparation of 4.3 reference substance solutions
Rhizoma alismatis control medicinal material 2g is taken, adds water 50ml, is heated to reflux 30 minutes, is let cool, filtered with absorbent cotton, filtrate oil
(60~90 DEG C) of ether shaking extraction 4 times, each 50ml (centrifuging when necessary), merges petroleum ether extract, is evaporated, residue adds oil
Ether 1ml makes dissolving.
The preparation of 4.4 negative samples
In prescription ratio and preparation process, the negative sample without Rhizoma Alismatis is prepared, feminine gender is prepared with method by " 4.2 "
Sample solution.
4.5 development system
Petroleum ether (60~90 DEG C)-chloroform-ethyl acetate (2：1：2).
4.6 color developing agent
5% phosphomolybdic acid ethanol solution.
The investigation of 4.7 thin-layer chromatography conditions
4.7.1 best point sample amount is investigated
4,6,8,9,12,14 μ l of test solution are drawn respectively, and 2,4,6,8,10,14 μ l of reference substance solution are put in not respectively
It on thin layer silica G plate, is investigated, is unfolded, taken out, dry, spray with color developing agent, 105 DEG C are heated 5min, ultraviolet in 365nm
It is inspected under lamp.See Figure 24, Figure 25.
By Figure 24, Figure 25 as it can be seen that 9~12 μ l point sample amount best results of test sample, when point sample amount is less than 9 μ l, test sample
Principal spot is unintelligible；Best 9~12 μ l of point sample amount of reference substance all may be used.
With reference to above-mentioned investigation experimental result, it may be determined that lamellae is silica G plate, and test sample and the best point sample amount of reference substance are
9~12 μ l.It is verified through preliminary experiment, determines the thin-layer identification method of rhizoma alismatis, and according to《Chinese Pharmacopoeia》2010 editions annex Ⅹ
VIII A quality standards in Chinese drugs analysis method verification guides principle carries out methodology validation.
4.8 methodology validation
4.8.1 specificity is investigated
It is releived capsule sample with the gout of three batches, carries out specificity experiment according to the above method, as a result see Figure 26.
As seen from Figure 26, negative sample is noiseless, and principal spot Rf values are about 0.48；With control medicinal material chromatography corresponding position
On, show the clear spot of same color, spot rounding, good separating effect.Show that this method specificity is strong, spot development is clear.
4.8.2 serviceability test
It is releived capsule sample, 3 different manufacturers lamellaes is compared respectively, 3 kinds of differences with the gout of three batches
Temperature and humidity are tested as variable according to above-mentioned discrimination method.
4.8.2.1 different manufacturers lamellae is tested, and as a result sees Figure 27-Figure 29.
3 different manufacturers durabilities the experimental results showed that, gout is releived the thin layer discriminating figure principal spot of desmodium in capsule
Separating effect does very well, spot rounding, and different manufacturers lamellae influences principal spot less, and repeatability is preferably.
2.8.2.2 different temperatures and humid test
Different temperatures and humidity set parameter are shown in Table 1.Experiment collection of illustrative plates is shown in Figure 30-Figure 32.
The investigation of 1 different temperatures of table and relative humidity
By the investigation experimental patterns of different temperatures and relative humidity as it can be seen that in test sample chromatography, with reference substance chromatography
On corresponding position, the fluorescence spot of same color is shown, clear spot, good separating effect, Rf values are moderate, and negative control is without dry
It disturbs.It follows that temperature and relative humidity change in usual environmental field, the validity of the discrimination method is not influenced.
The invention has the beneficial effects that：It is provided by the invention treatment acute gouty arthritis, gout Chinese medicine preparation
Detection method increase the assay project of desmodium, the root of Chinese clematis, the discriminating project of rhizoma alismatis and desmodium.Wherein desmodium
Content assaying method have easy to operate, at low cost, Check-Out Time is short, while has the good separating effect, sensitive, accurate etc. excellent
Point；Desmodium, the root of Chinese clematis, rhizoma alismatis discrimination method have that accuracy is high, precision is high, the rate of recovery is high, stability is high, specificity
By force, the advantages that repeatability is preferable, noiseless.The present invention can effectively detect whether product is qualified, whether quality is good and bad, ensure
The consistency of drug effect and the stability of quality, further ensure product quality stabilization and clinical application safely, have
Effect.
Description of the drawings
Fig. 1 is the system suitability figure that HPLC measures Kaempferol；
Fig. 2 is the HPLC figures of Kaempferol reference substance；
Fig. 3 is the HPL figures for lacking desmodium negative control sample；
Fig. 4 is the desmodium thin layer identification result figure with polyamide film lamellae；
Fig. 5 is the desmodium thin layer identification result figure with silica gel g thin-layer plate；
Fig. 6 is that desmodium differentiates that test sample point sample amount is respectively the thin-layer developing design sketch of 1,2,3,4,5 μ l from left to right；
Fig. 7 is the thin-layer developing design sketch that desmodium control medicinal material point sample amount is respectively from left to right 1,2,3,4,5 μ l；
Fig. 8 is that the specificity that desmodium thin layer differentiates investigates test effect figure；
Fig. 9 is the desmodium thin layer identification result figure of 1 lamellae of producer；
Figure 10 is the desmodium thin layer identification result figure of 2 lamellae of producer；
Figure 11 is the desmodium thin layer identification result figure of 3 lamellae of producer；
Figure 12 is the desmodium thin layer identification result figure under the conditions of 20 DEG C of temperature, humidity 50%；
Figure 13 is the desmodium thin layer identification result figure under the conditions of 25 DEG C of temperature, humidity 55%；
Figure 14 is the desmodium thin layer identification result figure under the conditions of 35 DEG C of temperature, humidity 70%；
In above-mentioned Fig. 4-5 and Fig. 8-14,1,2,3 be respectively that the gouts of 3 batches is releived capsule sample, and S is desmodium pair
According to medicinal material, S1 is to lack desmodium negative sample.
Figure 15 is that the root of Chinese clematis differentiates that test sample point sample amount is respectively the thin-layer developing of 1,2,3,5,7,10,11 μ l from left to right
Design sketch；
Figure 16 is the thin-layer developing effect that oleanolic acid reference substance point sample amount is respectively from left to right 1,2,3,4,5,6,7 μ l
Figure；
Figure 17 is that the specificity that root of Chinese clematis thin layer differentiates investigates test effect figure；
Figure 18 is the root of Chinese clematis thin layer identification result figure of 1 lamellae of producer；
Figure 19 is the root of Chinese clematis thin layer identification result figure of 2 lamellae of producer；
Figure 20 is the root of Chinese clematis thin layer identification result figure of 3 lamellae of producer；
Figure 21 is the root of Chinese clematis thin layer identification result figure under the conditions of 20 DEG C of temperature, humidity 50%；
Figure 22 is the root of Chinese clematis thin layer identification result figure under the conditions of 25 DEG C of temperature, humidity 55%；
Figure 23 is the root of Chinese clematis thin layer identification result figure under the conditions of 35 DEG C of temperature, humidity 70%；
In above-mentioned Figure 15-23,1,2,3 be respectively that the gouts of 3 batches is releived capsule sample, and S is oleanolic acid reference substance,
S1 is to lack root of Chinese clematis negative sample.
Figure 24 is that rhizoma alismatis differentiates that test sample point sample amount is respectively the thin-layer developing effect of 4,6,8,9,12,14 μ l from left to right
Figure；
Figure 25 is the thin-layer developing effect that rhizoma alismatis control medicinal material point sample amount is respectively from left to right 2,4,6,8,10,14 μ l
Figure；
Figure 26 is that the specificity that rhizoma alismatis thin layer differentiates investigates test effect figure；
Figure 27 is the rhizoma alismatis thin layer identification result figure of 1 lamellae of producer；
Figure 28 is the rhizoma alismatis thin layer identification result figure of 2 lamellae of producer；
Figure 29 is the rhizoma alismatis thin layer identification result figure of 3 lamellae of producer；
Figure 30 is the rhizoma alismatis thin layer identification result figure under the conditions of 20 DEG C of temperature, humidity 50%；
Figure 31 is the rhizoma alismatis thin layer identification result figure under the conditions of 25 DEG C of temperature, humidity 55%；
Figure 32 is the rhizoma alismatis thin layer identification result figure under the conditions of 35 DEG C of temperature, humidity 70%；
In above-mentioned Figure 24-32,1,2,3 be respectively that the gouts of 3 batches is releived capsule sample, and S is rhizoma alismatis control medicinal material, S1
To lack rhizoma alismatis negative sample.
Specific embodiment
The present invention is further introduced below in conjunction with specific embodiment.
It is of the present invention it is a kind of treat acute gouty arthritis, gout Chinese medicine preparation detection method, be not limited to
Lower embodiment.
Embodiment 1
Prescription：
Desmodium 300g root of Chinese clematis 150g Piper wallichii (Miq.) Hand.-Mazz.s 100g
Starflowerlike loosestrife root or herb 150g siphonostegia chinensis 150g rhizoma alismatis 120g
Asiatic plantain 100g
Preparation method：
More than seven, desmodium is extracted with 75% alcohol reflux, filtration, and recycling ethyl alcohol is simultaneously concentrated into appropriate.The dregs of a decoction and prestige
Remaining 6 taste such as medicine xiaolingxian, add water to cook three times, and every time, filtration, filtrate merges with above-mentioned alcoholic solution, and thick paste is concentrated under reduced pressure into, does
It is dry, crush, sieving, mix with appropriate amount of auxiliary materials, be sieved, mixing, loading capsule, be made 1000 to get.
The detection method of the Chinese medicine preparation is as follows：
(1) this product content 3g is taken, adds 80% methanol 50ml, is heated to reflux 1 hour, lets cool, is filtered, filtrate is evaporated, residual
Slag adds water 10ml to make dissolving, with ether shake medicine extract 2 times, each 10ml, discard ether solution, aqueous adds dilute hydrochloric acid 10ml, puts water
It heats 1 hour, takes out in bath, rapid to cool down, with ethyl acetate shaking extraction 2 times, each 20ml, combined ethyl acetate uses water
30ml is washed, and discards aqueous, acetic acid ethyl fluid is evaporated, and residue adds methanol 2ml to make dissolving, as test solution.Separately take money
Careless control medicinal material 0.5g, is made in the same way of control medicinal material solution.It is tested according to thin-layered chromatography (VI B of annex), draws test solution 3
μ l, 4 μ l of reference substance solution are put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid (10：8：1) it is expansion
Agent is unfolded, and takes out, dries, spray with 5% alchlor ethanol solution, is heated several minutes at 105 DEG C, puts ultraviolet lamp (365nm)
Under inspect.In test sample chromatography, on control medicinal material corresponding position, showing the fluorescence spot of same color.
(2) this product content 2g is taken, adds ethyl alcohol 50ml, is heated to reflux 2 hours, is filtered, filtrate is concentrated into 20ml, adds hydrochloric acid
3ml is heated to reflux 1 hour, adds water 10ml, let cool, and adds petroleum ether (60~90 DEG C) 25ml shakings extraction 4 times, each 25ml, stone
Oily ether is evaporated, and residue absolute ethyl alcohol 10ml dissolves, as test solution.Oleanolic acid reference substance separately is taken, adds absolute ethyl alcohol
Solution of the 1ml containing 0.45mg is made, as reference substance solution.It is tested according to thin-layered chromatography (VI B of annex), draws test solution
7 μ l, 3 μ l of reference substance solution are put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid (20：3：0.2) it is
Solvent, lamellae put presaturation 10 minutes in expansion cylinder, are unfolded, and take out, dry, spray with 10% ethanol solution of sulfuric acid, 105
It is clear DEG C to be heated to spot development.In test sample chromatography, on position corresponding with reference substance chromatography, the spot of same color is shown
Point.
Embodiment 2
Prescription：
Desmodium 300g root of Chinese clematis 150g Piper wallichii (Miq.) Hand.-Mazz.s 100g
Starflowerlike loosestrife root or herb 150g siphonostegia chinensis 150g rhizoma alismatis 120g
Asiatic plantain 100g
Preparation method：
More than seven, desmodium is extracted with 75% alcohol reflux, filtration, and recycling ethyl alcohol is simultaneously concentrated into appropriate.The dregs of a decoction and prestige
Remaining 6 taste such as medicine xiaolingxian, add water to cook three times, and every time, filtration, filtrate merges with above-mentioned alcoholic solution, and thick paste is concentrated under reduced pressure into, does
It is dry, crush, sieving, mix with appropriate amount of auxiliary materials, be sieved, mixing, loading capsule, be made 1000 to get.
The detection method of the Chinese medicine preparation is as follows
(1) this product content 2g is taken, adds ethyl alcohol 50ml, is heated to reflux 2 hours, is filtered, filtrate is concentrated into 20ml, adds hydrochloric acid
3ml is heated to reflux 1 hour, adds water 10ml, let cool, and adds petroleum ether (60~90 DEG C) 25ml shakings extraction 4 times, each 25ml, stone
Oily ether is evaporated, and residue absolute ethyl alcohol 10ml dissolves, as test solution.Oleanolic acid reference substance separately is taken, adds absolute ethyl alcohol
Solution of the 1ml containing 0.45mg is made, as reference substance solution.It is tested according to thin-layered chromatography (VI B of annex), draws test solution
7 μ l, 3 μ l of reference substance solution are put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid (20：3：0.2) it is
Solvent, lamellae put presaturation 10 minutes in expansion cylinder, are unfolded, and take out, dry, spray with 10% ethanol solution of sulfuric acid, 105
It is clear DEG C to be heated to spot development.In test sample chromatography, on position corresponding with reference substance chromatography, the spot of same color is shown
Point.
(2) this product content 10g is taken, adds ethyl alcohol 50ml, stirs evenly, is ultrasonically treated 90 minutes, filtration, filtrate is evaporated, residue
Water 50ml is added to make dissolving, filters off not tolerant, filtrate (60~90 DEG C) shaking extractions 4 times, each 50ml of petroleum ether merge oil
Ether liquid, is evaporated, and residue adds petroleum ether (60~90 DEG C) 1ml to make dissolving, as test solution.Rhizoma alismatis control medicinal material 2g separately is taken,
Add water 50ml, be heated to reflux 30 minutes, let cool, filtered with absorbent cotton, filtrate shakes extraction 4 times with petroleum ether, and each 50ml (must
Centrifuged when wanting), merge petroleum ether extract, be evaporated, residue adds petroleum ether (60~90 DEG C) 1ml to make dissolving, as control medicinal material
Solution.It is tested according to thin-layered chromatography (VI B of annex), draws each 9~12 μ l of above two solution, put respectively thin in same silica G
On laminate, with petroleum ether (60~90 DEG C)-chloroform-ethyl acetate (2：1：2) it is solvent, is unfolded, take out, dry, sprays
With 5% phosphomolybdic acid ethanol solution, it is clear to be heated to spot development at 110 DEG C.In test sample chromatography, with control medicinal material corresponding positions
It puts, shows the spot of same color.
Chromatographic condition is with system suitability using octadecylsilane chemically bonded silica as filler；With -0.4% phosphorus of methanol
Acid solution (50：50) it is mobile phase；Detection wavelength is 360nm.Number of theoretical plate is calculated by Kaempferol not less than 2500.
The preparation of reference substance solution takes Kaempferol reference substance appropriate, accurately weighed, adds 80% methanol that every 1ml is made containing kaempferia galamga
The solution of 20 μ g of element to get.
The preparation of test solution takes this product content about 1.5g, accurately weighed, puts in conical flask with cover, and precision adds in
80% methanol 50ml, close plug, weighed weight are heated to reflux 1 hour, let cool, then weighed weight, and less loss is supplied with 80% methanol
Weight shakes up, filtration.Precision measures subsequent filtrate 25ml, and precision adds in hydrochloric acid 5ml, puts heating hydrolysis 1 hour in 90 DEG C of water-baths,
Take out, it is rapid to cool down, be transferred in 50ml measuring bottles, with 80% methanol dilution to scale, shake up, filter, take subsequent filtrate to get.
Measuring method is accurate respectively to draw reference substance solution and each 10 μ l of test solution, injects liquid chromatograph, measures, i.e.,
.
Embodiment 3
Prescription：
Desmodium 300g root of Chinese clematis 150g Piper wallichii (Miq.) Hand.-Mazz.s 100g
Starflowerlike loosestrife root or herb 150g siphonostegia chinensis 150g rhizoma alismatis 120g
Asiatic plantain 100g
Preparation method：
More than seven, desmodium is extracted with 75% alcohol reflux, filtration, and recycling ethyl alcohol is simultaneously concentrated into appropriate.The dregs of a decoction and prestige
Remaining 6 taste such as medicine xiaolingxian, add water to cook three times, and every time, filtration, filtrate merges with above-mentioned alcoholic solution, and thick paste is concentrated under reduced pressure into, does
It is dry, crush, sieving, mix with appropriate amount of auxiliary materials, be sieved, mixing, loading capsule, be made 1000 to get.
The detection method of the Chinese medicine preparation is as follows
(1) this product content 3g is taken, adds 80% methanol 50ml, is heated to reflux 1 hour, lets cool, is filtered, filtrate is evaporated, residual
Slag adds water 10ml to make dissolving, with ether shake medicine extract 2 times, each 10ml, discard ether solution, aqueous adds dilute hydrochloric acid 10ml, puts water
It heats 1 hour, takes out in bath, rapid to cool down, with ethyl acetate shaking extraction 2 times, each 20ml, combined ethyl acetate uses water
30ml is washed, and discards aqueous, acetic acid ethyl fluid is evaporated, and residue adds methanol 2ml to make dissolving, as test solution.Separately take money
Careless control medicinal material 0.5g, is made in the same way of control medicinal material solution.It is tested according to thin-layered chromatography (VI B of annex), draws test solution 3
μ l, 4 μ l of reference substance solution are put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid (10：8：1) it is expansion
Agent is unfolded, and takes out, dries, spray with 5% alchlor ethanol solution, is heated several minutes at 105 DEG C, puts ultraviolet lamp (365nm)
Under inspect.In test sample chromatography, on control medicinal material corresponding position, showing the fluorescence spot of same color.
Chromatographic condition is with system suitability using octadecylsilane chemically bonded silica as filler；With -0.4% phosphorus of methanol
Acid solution (50：50) it is mobile phase；Detection wavelength is 360nm.Number of theoretical plate is calculated by Kaempferol not less than 2500.
The preparation of reference substance solution takes Kaempferol reference substance appropriate, accurately weighed, adds 80% methanol that every 1ml is made containing kaempferia galamga
The solution of 20 μ g of element to get.
The preparation of test solution takes this product content about 1.5g, accurately weighed, puts in conical flask with cover, and precision adds in
80% methanol 50ml, close plug, weighed weight are heated to reflux 1 hour, let cool, then weighed weight, and less loss is supplied with 80% methanol
Weight shakes up, filtration.Precision measures subsequent filtrate 25ml, and precision adds in hydrochloric acid 5ml, puts heating hydrolysis 1 hour in 90 DEG C of water-baths,
Take out, it is rapid to cool down, be transferred in 50ml measuring bottles, with 80% methanol dilution to scale, shake up, filter, take subsequent filtrate to get.
Measuring method is accurate respectively to draw reference substance solution and each 10 μ l of test solution, injects liquid chromatograph, measures, i.e.,
.
Embodiment 4
Prescription：
Desmodium 300g root of Chinese clematis 150g Piper wallichii (Miq.) Hand.-Mazz.s 100g
Starflowerlike loosestrife root or herb 150g siphonostegia chinensis 150g rhizoma alismatis 120g
Asiatic plantain 100g
Preparation method：
More than seven, desmodium is extracted with 75% alcohol reflux, filtration, and recycling ethyl alcohol is simultaneously concentrated into appropriate.The dregs of a decoction and prestige
Remaining 6 taste such as medicine xiaolingxian, add water to cook three times, and every time, filtration, filtrate merges with above-mentioned alcoholic solution, and thick paste is concentrated under reduced pressure into, does
It is dry, crush, sieving, mix with appropriate amount of auxiliary materials, be sieved, mixing, loading capsule, be made 1000 to get.Or with conventional method system
Obtain other preparations such as tablet, pill, electuary, oral liquid, spray, injection, suspension, microball preparation.
The detection method of the Chinese medicine preparation is as follows
(1) this product content 10g is taken, adds ethyl alcohol 50ml, stirs evenly, is ultrasonically treated 90 minutes, filtration, filtrate is evaporated, residue
Water 50ml is added to make dissolving, filters off not tolerant, filtrate (60~90 DEG C) shaking extractions 4 times, each 50ml of petroleum ether merge oil
Ether liquid, is evaporated, and residue adds petroleum ether (60~90 DEG C) 1ml to make dissolving, as test solution.Rhizoma alismatis control medicinal material 2g separately is taken,
Add water 50ml, be heated to reflux 30 minutes, let cool, filtered with absorbent cotton, filtrate shakes extraction 4 times with petroleum ether, and each 50ml (must
Centrifuged when wanting), merge petroleum ether extract, be evaporated, residue adds petroleum ether (60~90 DEG C) 1ml to make dissolving, as control medicinal material
Solution.It is tested according to thin-layered chromatography (VI B of annex), draws each 9~12 μ l of above two solution, put respectively thin in same silica G
On laminate, with petroleum ether (60~90 DEG C)-chloroform-ethyl acetate (2：1：2) it is solvent, is unfolded, take out, dry, sprays
With 5% phosphomolybdic acid ethanol solution, it is clear to be heated to spot development at 110 DEG C.In test sample chromatography, with control medicinal material corresponding positions
It puts, shows the spot of same color.
【Assay】It is measured according to high performance liquid chromatography (VI D of annex).
Chromatographic condition is with system suitability using octadecylsilane chemically bonded silica as filler；With -0.4% phosphorus of methanol
Acid solution (50：50) it is mobile phase；Detection wavelength is 360nm.Number of theoretical plate is calculated by Kaempferol not less than 2500.
The preparation of reference substance solution takes Kaempferol reference substance appropriate, accurately weighed, adds 80% methanol that every 1ml is made containing kaempferia galamga
The solution of 20 μ g of element to get.
The preparation of test solution takes this product content about 1.5g, accurately weighed, puts in conical flask with cover, and precision adds in
80% methanol 50ml, close plug, weighed weight are heated to reflux 1 hour, let cool, then weighed weight, and less loss is supplied with 80% methanol
Weight shakes up, filtration.Precision measures subsequent filtrate 25ml, and precision adds in hydrochloric acid 5ml, puts heating hydrolysis 1 hour in 90 DEG C of water-baths,
Take out, it is rapid to cool down, be transferred in 50ml measuring bottles, with 80% methanol dilution to scale, shake up, filter, take subsequent filtrate to get.
Measuring method is accurate respectively to draw reference substance solution and each 10 μ l of test solution, injects liquid chromatograph, measures, i.e.,
.
Claims (2)
1. it is a kind of treat acute gouty arthritis, gout Chinese medicine preparation detection method, it is characterised in that：The Chinese medicine preparation
By weight by 30 parts of desmodium, 15 parts of the root of Chinese clematis, 10 parts of Piper wallichii (Miq.) Hand.-Mazz., 15 parts of starflowerlike loosestrife root or herb, 15 parts of siphonostegia chinensis, 12 parts of rhizoma alismatis and
10 parts of Asiatic plantain is made of bulk pharmaceutical chemicals；
The preparation method of the Chinese medicine preparation filters for desmodium is taken to be extracted with 75% alcohol reflux, recycles ethyl alcohol and be concentrated into suitable
Amount, obtains the dregs of a decoction and alcoholic solution, the dregs of a decoction and the root of Chinese clematis, Piper wallichii (Miq.) Hand.-Mazz., starflowerlike loosestrife root or herb, siphonostegia chinensis, rhizoma alismatis and Asiatic plantain adds water to cook
Three times, it filters, filtrate merges with alcoholic solution, thick paste is concentrated under reduced pressure into, dry, pulverize, and is sieved, is mixed with auxiliary material, is sieved, and mixes
It is even, be packed into capsule, be made 1000 to get；
The detection method includes differentiating and the measure project of content；Wherein differentiate be to desmodium, the root of Chinese clematis, rhizoma alismatis thin layer
Chromatographic identification；Assay is the content with desmodium in Preparations by HPLC；The discrimination method tool of desmodium
Body is：The Chinese medicine preparation of 3 ~ 4g crude drug powders is taken, adds 80% 30 ~ 50ml of methanol, is heated to reflux 0.5 ~ 1 hour, lets cool, is filtered, filter
Liquid is evaporated, and residue adds 8 ~ 10ml of water to make dissolving, with ether shake medicine extract 2 ~ 4 times, 10 ~ 30ml, discards ether solution every time, and aqueous adds
8 ~ 10ml of dilute hydrochloric acid puts in water-bath and heats 0.5 ~ 1 hour, takes out, rapid to cool down, and shakes extraction 2 ~ 4 times with ethyl acetate, every time
20 ~ 30ml, combined ethyl acetate are washed with 30 ~ 50ml of water, discard aqueous, and acetic acid ethyl fluid is evaporated, and residue adds 1 ~ 2ml of methanol
Make dissolving, as test solution；Desmodium 0.5 ~ 1.5g of control medicinal material separately is taken, is made in the same way of control medicinal material solution；According to thin layer
Chromatography experiment draws test solution 3 ~ 4 μ l, 3 ~ 4 μ l of control medicinal material solution, is put respectively on same silica gel g thin-layer plate, with
Toluene-ethyl acetate-formic acid=10 ~ 8：8~6：1 ~ 0.5 is solvent, is unfolded, takes out, dry, spray molten with 5% alchlor ethyl alcohol
Liquid heats several minutes at 105 DEG C, puts and inspected under the ultraviolet lamp of 365nm；In test sample chromatography, with control medicinal material corresponding positions
It puts, shows the fluorescence spot of same color；The discrimination method of the root of Chinese clematis is specially：The medicine Chinese medicine preparation of 2 ~ 3g crude drug powders is taken,
Adding 30 ~ 50ml of ethyl alcohol, be heated to reflux 1 ~ 2 hour, filter, filtrate is concentrated into 15 ~ 20ml, adds 3 ~ 6ml of hydrochloric acid, it is heated to reflux 0.5 ~
1 hour, add 8 ~ 10ml of water, let cool, add in 60 ~ 90 DEG C of 15 ~ 25ml of petroleum ether and shake extraction 3 ~ 4 times, every time 20 ~ 25ml, oil
Ether is evaporated, and residue 7 ~ 10ml of absolute ethyl alcohol dissolves, as test solution；Oleanolic acid reference substance separately is taken, adds absolute ethyl alcohol
Solution of the 1ml containing 0.45mg is made, as reference substance solution；It is tested according to thin-layered chromatography, draws test solution 7 ~ 8 μ l, right
According to 2 ~ 3 μ l of product solution, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid=20 ~ 17：3~1.5：0.2~
0.1 is solvent, and lamellae puts presaturation 5 ~ 10 minutes in expansion cylinder, is unfolded, and takes out, dries, spray molten with 10% sulfuric acid ethyl alcohol
It is clear to be heated to spot development at 105 DEG C for liquid；In test sample chromatography, on position corresponding with reference substance chromatography, identical face is shown
The spot of color；The discrimination method of rhizoma alismatis is specially：The medicine Chinese medicine preparation of 8 ~ 10g crude drug powders is taken, adds 40 ~ 50ml of ethyl alcohol, stirs evenly,
It is ultrasonically treated 70 ~ 90 minutes, filtration, filtrate is evaporated, and residue adds 40 ~ 50ml of water to make dissolving, filters off not tolerant, and filtrate is with 60 ~ 90
DEG C petroleum ether shaking extraction 3 ~ 4 times, every time 40 ~ 50ml, merge petroleum ether liquid, be evaporated, residue adds 60 ~ 90 DEG C of petroleum ether
1ml makes dissolving, as test solution；Rhizoma alismatis 1.5 ~ 2g of control medicinal material separately is taken, adds 40 ~ 50ml of water, is heated to reflux 30 minutes, puts
It is cold, it is filtered with absorbent cotton, filtrate shakes extraction 3 ~ 4 times with petroleum ether, every time 30 ~ 50ml, merges petroleum ether extract, is evaporated,
Residue adds 60 ~ 90 DEG C of 0.5 ~ 1ml of petroleum ether to make dissolving, as control medicinal material solution；It tests, draws above-mentioned according to thin-layered chromatography
Each 9 ~ 12 μ l of two kinds of solution are put respectively on same silica gel g thin-layer plate, with 60 ~ 90 DEG C of petroleum ether-chloroform-acetic acid second
Ester=2 ~ 1：1~0.5：2 ~ 1 be solvent, is unfolded, takes out, dry, spray with 5% phosphomolybdic acid ethanol solution, spot is heated at 110 DEG C
Point colour developing is clear；In test sample chromatography, on control medicinal material corresponding position, showing the spot of same color；
Desmodium content assaying method is using Kaempferol reference substance as control, with -0.4% phosphoric acid solution=50 of methanol：50 be flowing
It is mutually the high performance liquid chromatography of mobile phase；The content assaying method of desmodium is specially：
The assay of Kaempferol in desmodium：
Chromatographic condition is with system suitability using octadecylsilane chemically bonded silica as filler；It is molten with -0.4% phosphoric acid of methanol
Liquid=50：50 be mobile phase；Detection wavelength is 360nm；Plate number is calculated by Kaempferol not less than 2500；
The preparation of reference substance solution；Take Kaempferol reference substance appropriate, it is accurately weighed, add 80% methanol that every 1ml is made containing Kaempferol 20
The solution of μ g to get；
The preparation of test solution；The medicine Chinese medicine preparation for being equivalent to 1.5g crude drug powders is taken, it is accurately weighed, put conical flask with cover
In, precision adds in 80% methanol 50ml, and close plug, weighed weight is heated to reflux 1 hour, lets cool, then weighed weight, with 80% methanol
The weight of less loss is supplied, is shaken up, is filtered；Precision measures subsequent filtrate 25ml, and precision adds in hydrochloric acid 5ml, puts in 90 DEG C of water-baths and heat
Hydrolysis 1 hour is taken out, rapid to cool down, and is transferred in 50ml measuring bottles, with 80% methanol dilution to scale, is shaken up, and is filtered, is taken continuous filter
Liquid to get；
Measuring method；It is accurate respectively to draw reference substance solution and each 10 μ l of test solution, inject liquid chromatograph, measure to get.
2. the detection method of the Chinese medicine preparation for the treatment of acute gouty arthritis according to claim 1, gout, feature
It is：The Chinese medicine preparation can be prepared into any conventional formulation, including tablet, pill, capsule, electuary, oral liquid,
Spray, injection, suspension, microball preparation.
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