CN106324117A - Quality detection method for dysuria remedying granules - Google Patents
Quality detection method for dysuria remedying granules Download PDFInfo
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Abstract
The invention discloses a quality detection method for Chinese patent medicine dysuria remedying granules for treating chronic prostatitis. The quality detection method comprises the steps that the thin layer chromatography is adopted for recognizing cinnamon, Chinese violets, radix scrophulariae, golden cypress, radix sophorae flavescentis, rhizoma dioscoreae hypoglaucae and radix cyathulae in the preparation, and meanwhile HPLC is adopted for determining the content of berberine hydrochloride of the golden cypress in the preparation and the content of amygdalin in peach kernels. The quality detection method is stable, reliable, high in specificity, good in reproducibility, capable of comprehensively and effectively controlling the quality of the dysuria remedying granules, and capable of stabilizing the product quality, ensuring the clinical medication safety and effectiveness and better meeting the requirement for medical treatment and market.
Description
Technical field
The present invention relates to a kind of Chinese patent medicine Longbi Xintong granule quality determining method treating chronic prostatitis, in belonging to
Medicine analysis of pharmaceutical dosage forms field.
Background technology
Chronic prostatitis (chronic prostatitis, CP) is urinary system andrology commonly encountered diseases, difficult disease, is apt to occur in green grass or young crops
Prime of life male.Its etiology and pathogenesis is complicated, have course of disease length, delay repeatedly, the feature such as symptom is various, clinic mainly shows as urinating
Abnormal, pelvis area pain or discomfort, even with sexual dysfunction etc..Though chronic prostatitis does not jeopardize patient vitals, but
Having a strong impact on physical and mental health and the quality of life of patient, reduce life Happiness Index, the highest medical expense is more given and is suffered from
Person family brings huge financial burden.
The cause of disease, the pathological change of primary disease are illustrated by modern medicine the most completely, the method that treatment also lacks specially good effect, still
Relying on antibiotic, physiotherapy etc., therapeutic effect is the most satisfied.In contrast to this, Chinese medicine uses organic conception, dialectical opinion
The feature controlled, carries out the side's of sending use of individuation targetedly in conjunction with many factors such as the body constitution of patient, feelings will, external environments
Medicine, with at many levels, Mutiple Targets is treated, thus has the advantage of uniqueness in terms of relief of symptoms, quality of making the life better, and obtains
Significant curative effect.Chinese medicine chronic prostatitis has become indispensable means during treatment CP.
Chinese patent CN1745808 discloses a kind of Chinese medicine composition treating chronic prostatitis and preparation method thereof,
This product has obtained III clinical trial phase at present, is declaring production official written reply, entitled Longbi Xintong granule.This product by
Cortex Phellodendri, Radix Sophorae Flavescentis, Herba Violae, Rhizoma Dioscoreae Hypoglaucae, Herba Dianthi, Radix Scrophulariae, Cortex Cinnamomi, Herba Lycopi, Pollen Typhae, Semen Persicae, Corium Erinacei Seu Hemiechianus (processing), Herba Salviae Plebeiae, river
Radix Achyranthis Bidentatae 13 taste Chinese prescription, the extracted Chinese traditional compound medicine being processed into, can immunotherapy targeted autoantibody merit chronic prostatitis.Infirmity
Close glad logical granule and pass through plurality of Chinese organic compatibility, treating both the principal and secondary aspects of a disease, can alleviate rapidly for symptom again for etiological treatment
Patient is ailing, than simple chemical medicine and biological preparation more advantage.And Chinese medicine is natural drug, toxic and side effects is little, clinical application
Safer.
Summary of the invention
The technical problem to be solved is on the basis of Chinese patent CN1745808, studies preparation further
Quality determining method.In order to control product quality fully and effectively, the present inventor, through substantial amounts of test, optimizes
Upgrade preparation Chinese crude drug and the method for qualitative and quantitative detection of medical material effective ingredient, established the matter that Chinese medicine Longbi Xintong granule is new
Quantity measuring method.
It is an object of the invention to provide the quality testing of a kind of Chinese patent medicine Longbi Xintong granule treating chronic prostatitis
Method, described Longbi Xintong granule be by Cortex Phellodendri, Radix Sophorae Flavescentis, Herba Violae, Rhizoma Dioscoreae Hypoglaucae, Herba Dianthi, Radix Scrophulariae, Cortex Cinnamomi, Herba Lycopi, Pollen Typhae,
Semen Persicae, Corium Erinacei Seu Hemiechianus (processing), Herba Salviae Plebeiae, Radix Cyathulae 13 taste Chinese prescription, extracted be processed into.This quality determining method is
Differentiate Cortex Cinnamomi, Herba Violae, Radix Scrophulariae, Cortex Phellodendri, Radix Sophorae Flavescentis, Rhizoma Dioscoreae Hypoglaucae and Radix Cyathulae in preparation with thin layer chromatography, use simultaneously
HPLC measures the amygdaloside content in the content of berberine hydrochloride of Cortex Phellodendri in preparation and Semen Persicae, thus realizes to the difficulty in urination glad logical
Grain quality is evaluated all sidedly and controls, and provides comprehensively for the real and fake discrimination of Longbi Xintong granule and inherent quality detection, can
The foundation leaned on, it is ensured that the stability of product quality and the safety of clinical application, effectiveness.
Technical solution of the present invention is with Cortex Cinnamomi, Herba Violae, Radix Scrophulariae, Cortex Phellodendri, hardship in indentification by TLC Longbi Xintong granule
Ginseng, Rhizoma Dioscoreae Hypoglaucae and Radix Cyathulae, specifically include the following step:
1, Cortex Cinnamomi differentiates
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking,
Filtering, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with dilute hydrochloric acid adjust pH value to 1-2, with ether extraction 2 times, each 20mL,
Merging ether solution, low temperature volatilizes, and residue adds methanol 2mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: take cinnamic aldehyde reference substance and add ethanol and make every 1mL containing the solution of 1 μ L, as comparison
Product solution；
3) point sample, expansion: draw need testing solution 2-5 μ L, reference substance solution 2 μ L, put respectively in same silica gel g thin-layer plate
On, with 60-90 DEG C of petroleum ether: ethyl acetate=17:3, as developing solvent, launches, and takes out, dries, and sprays and tries with dinitrophenylhydrazine ethanol
Liquid；In test sample chromatograph, on position corresponding with reference substance chromatograph, the speckle of aobvious same color.
2, Herba Violae differentiates
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking,
Filtering, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with dilute hydrochloric acid adjust pH value to 1-2, with ether extraction 2 times, each 20mL,
Merging ether solution, low temperature volatilizes, and residue adds methanol 2mL makes dissolving, as need testing solution；
2) preparation of control medicinal material solution: separately take Herba Violae control medicinal material 2g, adds methanol 20mL, supersound extraction 20 points
Clock, filters, is evaporated, and residue adds hot water 20mL makes dissolving, filters, with dilute hydrochloric acid adjust pH value to 1-2, with ether extraction 2 times, every time
20mL, merges ether solution, volatilizes, and residue adds methanol 2mL makes dissolving, as control medicinal material solution；
3) point sample, expansion: draw above two solution each 2-5 μ L, put respectively on same silica gel g thin-layer plate, with trichlorine
Methane: acetone: formic acid=25:20:4 is developing solvent, launches, and takes out, dries, put and inspect under 365nm ultra-violet lamp；Test sample color
In spectrum, on position corresponding with control medicinal material chromatograph, the fluorescence principal spot of aobvious same color.
3, Radix Scrophulariae differentiates
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking,
Filtering, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with dilute hydrochloric acid adjust pH value to 1-2, with ether extraction 2 times, each 20mL,
Water liquid after extraction ethyl acetate is extracted 3 times, each 20mL, and combined ethyl acetate solution is evaporated, and residue is by a small amount of 50% first
Alcohol dissolves, and is added on 100-200 mesh neutral alumina column, and with 50% methanol 30mL eluting, eluent is evaporated, and residue adds methanol
0.5mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: take Radix Scrophulariae control medicinal material 1g, adds methanol 20mL, supersound extraction 30 minutes, filters, steam
Dry, the residue 20mL that adds water makes dissolving, extracts 2 times by ethyl acetate, each 20mL, combined ethyl acetate liquid, is evaporated, and residue adds first
Alcohol 1mL makes dissolving, as control medicinal material solution；
3) point sample, expansion: draw need testing solution 5-10 μ L, control medicinal material solution 4 μ L, put in same silica gel G thin respectively
On laminate, with chloroform: methanol: water: lower floor's solution of formic acid=12:4:1:1, as developing solvent, launches, and takes out, dries, spray
With 5% vanillin-sulfuric acid solution, 105 DEG C to be heated to spot development clear；In test sample chromatograph, corresponding to control medicinal material chromatograph
Position on, the speckle of aobvious same color.
4, Cortex Phellodendri differentiates
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking,
Filtering, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with dilute hydrochloric acid adjust pH value to 1-2, with ether extraction 2 times, each 20mL,
Water liquid after extraction with ethyl acetate extract 3 times, each 20mL, the water liquid after extraction with ammonia solution adjust pH value to 10-11, with three
Chloromethanes 20mL extracts 1 time, and chloroform liquid is evaporated, and residue adds methanol 2mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: take berberine hydrochloride reference substance, adds methanol and makes every 1mL solution containing 0.5mg, make
For reference substance solution；
3) point sample, expansion: draw above two solution each 1-2 μ L, put respectively on same silica gel g thin-layer plate, with positive fourth
Alcohol: glacial acetic acid: water=7:1:2 is developing solvent, launches, and takes out, dries, put and inspect under 365nm uviol lamp；In test sample chromatograph,
On position corresponding with reference substance chromatograph, the fluorescence speckle of aobvious same color.
5, Radix Sophorae Flavescentis differentiates
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking,
Filtering, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with dilute hydrochloric acid adjust pH value to 1-2, with ether extraction 2 times, each 20mL,
Water liquid after extraction with ethyl acetate extract 3 times, each 20mL, the water liquid after extraction with ammonia solution adjust pH value to 10-11, with three
Chloromethanes 20mL extracts 1 time, and chloroform liquid is evaporated, and residue adds methanol 2mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: take matrine reference substance, adds methanol and makes every 1mL solution containing 0.2mg, as right
According to product solution；
3) point sample, expansion: draw need testing solution 2-5 μ L, reference substance solution 5 μ L, put respectively in same silica gel G thin layer
On plate, with chloroform: methanol: lower floor's solution of ammonia=5:0.6:0.2, as developing solvent, launches, and takes out, dries, and spray is to change
Good bismuth potassium iodide test solution；In test sample chromatograph, on position corresponding with reference substance chromatograph, the speckle of aobvious same color.
6, Rhizoma Dioscoreae Hypoglaucae differentiates
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking,
Filtering, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with dilute hydrochloric acid adjust pH value to 1-2, with ether extraction 2 times, each 20mL,
Water liquid after extraction with ethyl acetate extract 3 times, each 20mL, the water liquid after extraction with ammonia solution adjust pH value to 10-11, with three
Chloromethanes 20mL extracts 1 time, takes the water liquid 10mL after extraction, extracts 2 times by ethyl acetate, each 10mL, discards ethyl acetate
Liquid, water liquid water saturated n-butanol extraction 2 times, each 10mL, merge n-butanol layer, wash 1 time with ammonia solution 20mL, discard
Ammonia layer, n-butanol layer water bath method, residue adds methanol 2mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: taking Rhizoma Dioscoreae Hypoglaucae control medicinal material 0.5g, add water 30mL, boils 10 minutes, lets cool, filter
Crossing, the saturated n-butanol extraction of filtrate water 2 times, each 20mL, merge n-butyl alcohol liquid, be evaporated, residue adds methanol 2mL to be made molten
Solve, as control medicinal material solution；
3) point sample, expansion: draw each 5 μ L of above two solution, put respectively on same silica gel g thin-layer plate, with three chloromethanes
Alkane: methanol: lower floor's solution of water=13:7:2 is developing solvent, launches, and takes out, dries, and sprays with 10% ethanol solution of sulfuric acid,
105 DEG C are heated to spot development clearly, put respectively and inspect under daylight and 365nm ultra-violet lamp；In test sample chromatograph, with compare
On the corresponding position of medical material chromatograph, the speckle of aobvious same color or fluorescence speckle.
7, Radix Cyathulae differentiates
1) preparation of need testing solution: take this product 16g, finely ground, add methanol 50mL supersound process 30 minutes, time add shaking,
Filtering, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with ether extraction twice, each 30mL, discards ether solution, and water liquid is with dilute
Hydrochloric acid adjusts pH value to 1-2, extracts twice by ethyl acetate, and each 20mL, acetic acid ethyl fluid is evaporated, residue methanol: acetic acid second
Ester=1:1 dissolves in right amount, is added on 100-200 mesh neutral alumina column, with methanol: the solution 40mL of ethyl acetate=1:1 washes
De-, to collect eluent, be evaporated, residue adds methanol 1mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: separately take Radix Cyathulae control medicinal material 2g, add methanol 50mL, is heated to reflux 1 hour, filter
Crossing, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with ether extraction twice, each 30mL, discards ether solution, the dilute salt of water liquid
Acid adjusts pH value to 1-2, extracts twice by ethyl acetate, and each 20mL, acetic acid ethyl fluid is evaporated, residue methanol: ethyl acetate
=1:1 dissolves in right amount, is added on 100-200 mesh neutral alumina column, with methanol: the solution 40mL eluting of ethyl acetate=1:1,
Collecting eluent, be evaporated, residue adds methanol 1mL makes dissolving, as reference substance solution；
3) point sample, expansion: draw each 5 μ L of above two solution, put respectively on same silica gel g thin-layer plate, with three chloromethanes
Alkane: methanol: formic acid=10:1.5:2 is developing solvent, launches, and takes out, dries, and spray, with 10% ethanol solution of sulfuric acid, adds at 105 DEG C
Heat is clear to spot development, puts and inspects under 365nm ultra-violet lamp；In test sample chromatograph, in position corresponding with control medicinal material chromatograph
Put, the fluorescence speckle of aobvious same color.
The present invention use in high effective liquid chromatography for measuring Longbi Xintong granule simultaneously the content of berberine hydrochloride of Cortex Phellodendri and
The content of the amygdaloside of Semen Persicae, comprises the following steps:
1, the berberine hydrochloride content of Cortex Phellodendri
1) preparation of need testing solution: take Longbi Xintong granule appropriate, finely ground, take 2g, accurately weighed, put in conical flask,
The accurate hydrochloric acid that adds: the solution 50mL of methanol=1:100, weighed weight, supersound process 30 minutes, lets cool, weighed weight, uses salt
Acid: methanol=1:100 solution supplies less loss weight, shakes up, filters, accurate absorption subsequent filtrate 3mL, is added in 100-200 mesh neutral
On alumina column, with methanol 50mL eluting, collecting eluent, water bath method, residue dissolves with methanol and constant volume is in 10mL capacity
In Ping, shake up, filter, to obtain final product；
2) preparation of reference substance solution: weigh berberine hydrochloride reference substance appropriate, adds methanol and makes every 1mL containing 0.03mg's
Solution, shakes up, and to obtain final product；
3) HPLC chromatogram condition: octadecylsilane chemically bonded silica is filler；Acetonitrile: 0.05mol/L sodium dihydrogen phosphate
Solution=30:70 is flowing phase；Detection wavelength is 346nm, and number of theoretical plate is calculated by berberine hydrochloride peak should be not less than 4000；
4) assay method: precision draws reference substance solution and need testing solution 10uL respectively, injects high performance liquid chromatograph,
According to step 3) described chromatographic condition mensuration, to obtain final product.
2, the amygdaloside assay of Semen Persicae
1) preparation of need testing solution: take Longbi Xintong granule appropriate, finely ground, take 5g, accurately weighed, put tool plug conical flask
In, accurate addition water 50mL, weighed weight, supersound process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with water,
Shaking up, centrifugal, accurate Aspirate supernatant 10mL puts in separatory funnel, extracts 2 times by ethyl acetate, each 20mL, discards acetic acid
Ethyl ester liquid, water liquid is put in evaporating dish, is washed with a small amount separatory funnel, is incorporated in the lump in evaporating dish, and water-bath is waved to without acetic acid
Ethyl ester taste, lets cool, and is transferred in 25mL measuring bottle, constant volume, shakes up, to obtain final product；
2) preparation of reference substance solution: weigh amygdaloside reference substance appropriate, adds methanol and makes molten containing 0.1mg of every 1mL
Liquid, shakes up, and to obtain final product；
3) HPLC chromatogram condition: octadecylsilane chemically bonded silica is filler；Methanol: water=18:82 is flowing phase；Inspection
Survey wavelength is 210nm, and number of theoretical plate is calculated by amygdaloside should be not less than 4000；
4) assay method: precision draws reference substance solution and need testing solution 5uL respectively, injects high performance liquid chromatograph,
According to step 3) described chromatographic condition mensuration, to obtain final product.
The present invention, on the basis of the glad logical proper mass detection method of the difficulty in urination, through optimizing and upgrading, establishes the difficulty in urination glad logical
The new method of granular mass detection.The method use thin layer chromatography respectively to Cortex Cinnamomi in preparation, Herba Violae, Radix Scrophulariae, Cortex Phellodendri,
Radix Sophorae Flavescentis, Rhizoma Dioscoreae Hypoglaucae and Radix Cyathulae carry out qualitative identification, reach multicomponent and jointly control, and utilize in HPLC quantitative determination preparation simultaneously
The content of berberine hydrochloride of Cortex Phellodendri and the content of the amygdaloside of Semen Persicae, it is possible to the most effectively control the matter of Longbi Xintong granule
Amount, it is achieved the quality of the pharmaceutical preparations is evaluated all sidedly, thus ensure that the stability of product quality and clinical use to greatest extent
The safety of medicine, effectiveness.Detection method of the present invention is scientific and reasonable, and condition is controlled, and specificity is strong, and stability is high,
There is the strongest practicality；The application of this quality determining method, it can be ensured that the clinical efficacy of Longbi Xintong granule, the most satisfied
Medical treatment and the needs in market.
The inventive method optimization and upgrading Longbi Xintong granule every discriminating of former detection method, decreases making of harmful reagent
With, and add the discriminating of Rhizoma Dioscoreae Hypoglaucae and Radix Cyathulae.Compared with initial quality detection method, Longbi Xintong granule quality of the present invention
Detection method has and following improves significantly:
1) the former thin-layer identification method of Cortex Cinnamomi uses aether backflow to extract, and owing to Cortex Cinnamomi volatile oil uses cyclodextrin inclusion compound, carries
Taking inefficient, this revision uses ether extraction after using methanol extraction again, and extraction ratio is higher, launches by primary colors spectral condition, aobvious
Color, negative noiseless, method specificity is good；
2) the former thin-layer identification method of Herba Violae is loaded down with trivial details, after new method is with methanol extraction, then with ether extraction, composes by primary colors
Condition is launched, and separates good, negative noiseless；
3) the former thin-layer identification method of Radix Scrophulariae 75% ethanol extraction, the sample impurity obtained is too many, and subsequent treatment is cumbersome,
Launching the separation of rear impurity interference Radix Scrophulariae, method cannot be reappeared.This revision methanol extraction, ether remove impurity, ethyl acetate carries
Removing impurity with aluminium oxide again after taking, method is simple.Launch still cannot efficiently separate with former developing solvent, change pharmacopeia Radix Scrophulariae medical material
Developing solvent after, the separation of the berberine smearing effects Radix Scrophulariae of top, after developing solvent adds appropriate formic acid, separate good,
Negative noiseless；
4) Cortex Phellodendri, Radix Sophorae Flavescentis are after methanol extraction, after ether, ethyl acetate defat, then by chloroform extraction, more former method
The chromatograph obtained is cleaner, negative noiseless；
5) Rhizoma Dioscoreae Hypoglaucae is mainly composed of saponin component, after methanol extraction, anti-with ether, ethyl acetate, chloroform
After multiple remove impurity, then wash with ammonia solution after n-butanol extraction, select the chromatographic condition of pharmacopeia with under chloroform-methanol-water
Layer solution is that developing solvent launches, and ethanol solution of sulfuric acid develops the color, clear spot, separates good, negative noiseless；
6) after Radix Cyathulae methanol extraction, ether remove impurity, after oxidized aluminum purifies, is launched by pharmacopeia chromatographic condition, top
The separation of berberine hangover interference Radix Cyathulae, adjust developing solvent ratio, and after adding a small amount of formic acid, top berberine speckle circle
Whole without hangover, do not disturb the separation of Radix Cyathulae.Owing to the cyasterone content in Radix Cyathulae is relatively low, and technique uses water to carry, and takes
Sample amount is adjusted to 16g, is equivalent to Radix Cyathulae medical material 9.6g, differentiates substantially, negative noiseless.
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of Longbi Xintong granule Cortex Phellodendri berberine hydrochloride, is for hydrochloric acid the most successively
Berberine reference substance, negative control chromatogram, test sample chromatogram；
Fig. 2 is the high-efficient liquid phase chromatogram of Longbi Xintong granule Semen Persicae amygdaloside, is followed successively by amygdaloside from top to bottom
Reference substance, negative control chromatogram, test sample chromatogram；
Fig. 3 is the thin-layer chromatogram of Cortex Cinnamomi in Longbi Xintong granule, and by order from left to right, wherein 1,4 is cinnamic aldehyde
Reference substance, 2 is sample, and 3 is the negative formulation samples in misrun osmanthus；
Fig. 4 is the thin-layer chromatogram of Herba Violae in Longbi Xintong granule, and by order from left to right, wherein 1,4 is purple
Flower Herba Violae control medicinal material, 2 is sample, and 3 is the negative formulation samples in misrun osmanthus；
Fig. 5 is the thin-layer chromatogram of Radix Scrophulariae in Longbi Xintong granule, and by order from left to right, wherein 1,4 is Radix Scrophulariae pair
According to medical material, 2 is the negative formulation samples lacking Radix Scrophulariae, and 3 is sample；
Fig. 6 is the thin-layer chromatogram of Cortex Phellodendri in Longbi Xintong granule, by order from left to right, wherein 1,4 hydrochloric acid Radix Berberidis Amurensis
Alkali reference substance, 2 is sample, and 3 is the negative formulation samples lacking Cortex Phellodendri；
Fig. 7 is the thin-layer chromatogram of Radix Sophorae Flavescentis in Longbi Xintong granule, and by order from left to right, wherein 1,4 is matrine
Reference substance, 2 is sample, 3 be sample be lack Radix Sophorae Flavescentis negative formulation samples；
Fig. 8 is the thin-layer chromatogram (inspecting under daylight) of Rhizoma Dioscoreae Hypoglaucae in Longbi Xintong granule, by order from left to right, its
In 1,4 be Rhizoma Dioscoreae Hypoglaucae control medicinal material, 2 is sample, and 3 is the negative formulation samples lacking Rhizoma Dioscoreae Hypoglaucae；
Fig. 9 is the thin-layer chromatogram (inspecting under 365nm ultra-violet lamp) of Rhizoma Dioscoreae Hypoglaucae in Longbi Xintong granule, and wherein 1,4 are
Rhizoma Dioscoreae Hypoglaucae control medicinal material, 2 is sample, and 3 is the negative formulation samples lacking Rhizoma Dioscoreae Hypoglaucae；
Figure 10 is the thin-layer chromatogram of Radix Cyathulae in Longbi Xintong granule, and by order from left to right, wherein 1,4 is river cattle
Knee joint control medicinal material, 2 is the negative formulation samples lacking Radix Cyathulae, and 3 is sample.
Detailed description of the invention
The HPLC methodological study of embodiment 1 berberine hydrochloride
1) instrument and reagent: Agilent 1100 high performance liquid chromatograph, VWD detector.Acetonitrile is chromatographically pure, remaining examination
Agent is analytical pure, and water is double distilled water.
2) chromatographic condition: Kromasil C18Chromatographic column (4.6mm × 250mm, 5 μm)；Flowing is acetonitrile: 0.05mol/L mutually
Sodium dihydrogen phosphate (including the triethylamine of 0.5%, with phosphorus acid for adjusting pH value to 3.0)=30:70；Detection wavelength is 346nm；Flow velocity
1mL/min.Number of theoretical plate is calculated by berberine hydrochloride peak should be not less than 3000.
3) preparation of need testing solution: take this product under content uniformity item, finely ground, take 2g, accurately weighed, put tool plug taper
In Ping, the accurate hydrochloric acid that adds: methanol=1:100 solution 50mL, weighed weight, supersound process 30 minutes, let cool, more weighed heavy
Amount, with hydrochloric acid: methanol=1:100 solution supplies the weight of less loss, shakes up, filters, accurate absorption subsequent filtrate 5mL, is added on neutrality
On alumina column (dry column-packing, with 30mL methanol prewashing for 100-200 mesh, 6g, internal diameter 1.2cm), with methanol 50mL eluting, wash
De-liquid is evaporated, and dissolves with reference to methanol, and constant volume is in 10mL measuring bottle, shakes up, filter, to obtain final product.
4) preparation of reference substance solution: it is appropriate that precision weighs berberine hydrochloride reference substance, adds methanol and makes every 1mL and contain
The solution of 0.05mg, to obtain final product.
5) preparation of negative control solution: take the negative control without Cortex Phellodendri medical material, according under the preparation of need testing solution
Operate with method, make negative control solution, from chromatogram it can be seen that noiseless in berberine hydrochloride position.See Fig. 1.
6) linear relationship is investigated: it is appropriate that precision weighs berberine hydrochloride reference substance, adds methanol and is made into the molten of 0.0554mg/mL
Liquid, is measured under above-mentioned chromatographic condition, respectively sample introduction 1 μ L, 2 μ L, 4 μ L, 6 μ L, 8 μ L, 10 μ L, measures peak area, such as table
1.To record peak area as vertical coordinate, reference substance sample size (μ g) is abscissa, draws standard curve, obtains regression equation Y=
4299.632X-21.704, r=0.99991, berberine hydrochloride sample size is good in 0.0554～0.554 μ g range internal linear.
Result investigated by table 1 linear relationship
7) precision test: accurate reference substance solution of drawing, repetition sample introduction 6 times, each 5 μ L, peak area RSD=
0.397%, show that instrument precision is good.The results are shown in Table 2.
Table 2 Precision test result
8) stability test: take same sample solution, 0h, 2h, 4h, 6h, 8h after preparation, measure, result shows
Sample is stable in 8h.The results are shown in Table 3.
Table 3 stability test result
9) replica test: take 6 parts of the sample of same lot number respectively, according to the sample preparation methods behaviour under " sample determination " item
Making, measure respectively, calculate content, RSD is 0.96%.The results are shown in Table 4.
Table 4 replica test result
10) recovery test: 6 parts of the sample of accurately weighed known content, is separately added into reference substance appropriate, with " test sample
The preparation of solution " preparation method under item measures, obtain berberine hydrochloride average recovery rate be %, RSD be 0.594%.Result is shown in
Table 5.
Table 5 recovery test result
Embodiment 2 amygdaloside HPLC methodological study
1) instrument and reagent: Agilent 1100 high performance liquid chromatograph, VWD detector.Methanol is chromatographically pure, remaining examination
Agent is analytical pure, and water is double distilled water.
2) chromatographic condition: Kromasil C18Chromatographic column (4.6mm × 250mm, 5 μm)；Flowing is methanol mutually: water=18:
82；Detection wavelength is 210nm；Flow velocity 0.8mL/min.Number of theoretical plate is calculated by amygdaloside peak should be not less than 3000.
3) preparation of need testing solution: take this product under content uniformity item, finely ground, take 1g, accurately weighed, put tool plug taper
In Ping, accurate addition water 50mL, weighed weight, supersound process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with water
Amount, shakes up, and centrifugal, precision measures supernatant 25mL and puts in separatory funnel, extracts 2 times by ethyl acetate, each 30mL, discards second
Acetoacetic ester, water liquid is transferred in evaporating dish, and separatory funnel is washed with a small amount again, is incorporated in evaporating dish, steam to without acetic acid second
Ester taste, is transferred to water liquid in 50mL measuring bottle, then is washed with a small amount evaporating dish, is incorporated in 50mL measuring bottle, adds water to scale,
Shake up, to obtain final product.
4) preparation of reference substance solution: it is appropriate that precision weighs amygdaloside reference substance, adds 50% methanol and makes every 1mL and contain
The solution of 0.05mg, to obtain final product.
5) preparation of negative control solution: take the negative control without Semen Persicae medical material, according under the preparation of need testing solution
Operate with method, make negative control solution, from chromatogram it can be seen that noiseless in amygdaloside position.See Fig. 2.
6) linear relationship is investigated: it is appropriate that precision weighs amygdaloside reference substance, adds 10% methanol and is made into 0.1085mg/mL's
Solution, is measured under above-mentioned chromatographic condition, respectively sample introduction 1 μ L, 2 μ L, 4 μ L, 6 μ L, 8 μ L, 10 μ L, measures peak area, as
Table 6.To record peak area as vertical coordinate, reference substance sample size (μ g) is abscissa, draws standard curve, obtains regression equation Y=
1254.987X-7.566, r=0.99994, paeonol sample size is good in 0.1085～1.085 μ g range internal linear.
Result investigated by table 6 linear relationship
7) precision test: accurate reference substance solution of drawing, repetition sample introduction 6 times, each 5 μ L, peak area RSD=
1.319%, show that instrument precision is good.The results are shown in Table 7.
Table 7 Precision test result
8) stability test takes same sample solution, 0h, 2h, 4h, 6h, 8h after preparation, measures, and result shows sample
Product are stable in 8h.The results are shown in Table 8.
Table 8 stability test result
9) replica test: take 6 parts of the sample of same lot number respectively, according to the sample preparation methods behaviour under " sample determination " item
Making, measure respectively, calculate content, RSD is 0.922%.The results are shown in Table 9.
Table 9 replica test result
10) recovery test: 6 parts of the sample of accurately weighed known content, is separately added into reference substance appropriate, with " test sample
The preparation of solution " preparation method under item measures, and obtaining amygdaloside average recovery rate is 99.09%, and RSD is 0.522%.Result
It is shown in Table 10.
Table 10 recovery test result
Cortex Cinnamomi indentification by TLC in embodiment 3 Longbi Xintong granule
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking,
Filtering, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with dilute hydrochloric acid adjust pH value to 1-2, with ether extraction 2 times, each 20mL,
Merging ether solution, low temperature volatilizes, and residue adds methanol 2mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: take cinnamic aldehyde reference substance and add ethanol and make every 1mL containing the solution of 1 μ L, as comparison
Product solution；
3) point sample, expansion: draw need testing solution 2-5 μ L, reference substance solution 2 μ L, put respectively in same silica gel g thin-layer plate
On, with 60-90 DEG C of petroleum ether: ethyl acetate=17:3, as developing solvent, launches, and takes out, dries, and sprays and tries with dinitrophenylhydrazine ethanol
Liquid；In test sample chromatograph, on position corresponding with reference substance chromatograph, the speckle of aobvious same color.
4) result: Fig. 3 is the thin-layer chromatogram of Cortex Cinnamomi in Longbi Xintong granule, and result shows: the sample containing Cortex Cinnamomi exists
On the position corresponding with cinnamic aldehyde reference substance chromatograph, the speckle of aobvious same color.Chromatographic isolation is good, and the characteristic spots of Cortex Cinnamomi is dashed forward
Going out, the TLC method of foundation can be as the quality determining method of Cortex Cinnamomi in Longbi Xintong granule.
Herba Violae indentification by TLC in embodiment 4 Longbi Xintong granule
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking,
Filtering, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with dilute hydrochloric acid adjust pH value to 1-2, with ether extraction 2 times, each 20mL,
Merging ether solution, low temperature volatilizes, and residue adds methanol 2mL makes dissolving, as need testing solution；
2) preparation of control medicinal material solution: separately take Herba Violae control medicinal material 2g, adds methanol 20mL, supersound extraction 20 points
Clock, filters, is evaporated, and residue adds hot water 20mL makes dissolving, filters, with dilute hydrochloric acid adjust pH value to 1-2, with ether extraction 2 times, every time
20mL, merges ether solution, volatilizes, and residue adds methanol 2mL makes dissolving, as control medicinal material solution；
3) point sample, expansion: draw above two solution each 2-5 μ L, put respectively on same silica gel g thin-layer plate, with trichlorine
Methane: acetone: formic acid=25:20:4 is developing solvent, launches, and takes out, dries, put and inspect under 365nm ultra-violet lamp；Test sample color
In spectrum, on position corresponding with control medicinal material chromatograph, the fluorescence principal spot of aobvious same color.
4) result: Fig. 4 is the thin-layer chromatogram of Herba Violae in Longbi Xintong granule, and result shows: containing Herba Violae
Sample on the position corresponding with Herba Violae control medicinal material chromatograph, the speckle of aobvious same color.Chromatographic isolation is good, pale reddish brown
The characteristic spots of Herba Violae highlights, and the TLC method of foundation can be as the quality determining method of Herba Violae in Longbi Xintong granule.
Radix Scrophulariae indentification by TLC in embodiment 5 Longbi Xintong granule
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking,
Filtering, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with dilute hydrochloric acid adjust pH value to 1-2, with ether extraction 2 times, each 20mL,
Water liquid after extraction ethyl acetate is extracted 3 times, each 20mL, and combined ethyl acetate solution is evaporated, and residue is by a small amount of 50% first
Alcohol dissolves, and is added on 100-200 mesh neutral alumina column, and with 50% methanol 30mL eluting, eluent is evaporated, and residue adds methanol
0.5mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: take Radix Scrophulariae control medicinal material 1g, adds methanol 20mL, supersound extraction 30 minutes, filters, steam
Dry, the residue 20mL that adds water makes dissolving, extracts 2 times by ethyl acetate, each 20mL, combined ethyl acetate liquid, is evaporated, and residue adds first
Alcohol 1mL makes dissolving, as control medicinal material solution；
3) point sample, expansion: draw need testing solution 5-10 μ L, control medicinal material solution 4 μ L, put in same silica gel G thin respectively
On laminate, with chloroform: methanol: water: lower floor's solution of formic acid=12:4:1:1, as developing solvent, launches, and takes out, dries, spray
With 5% vanillin-sulfuric acid solution, 105 DEG C to be heated to spot development clear；In test sample chromatograph, corresponding to control medicinal material chromatograph
Position on, the speckle of aobvious same color.
4) result: Fig. 5 is the thin-layer chromatogram of Radix Scrophulariae in Longbi Xintong granule, and result shows: the sample containing Radix Scrophulariae exists
On the position corresponding with Radix Scrophulariae control medicinal material chromatograph, the speckle of aobvious same color.Chromatographic isolation is good, and the characteristic spots of Radix Scrophulariae is dashed forward
Going out, the TLC method of foundation can be as the quality determining method of Radix Scrophulariae in Longbi Xintong granule.
In embodiment 6 Longbi Xintong granule, Analysis of Cortex Phellodendri By Tlc differentiates
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking,
Filtering, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with dilute hydrochloric acid adjust pH value to 1-2, with ether extraction 2 times, each 20mL,
Water liquid after extraction with ethyl acetate extract 3 times, each 20mL, the water liquid after extraction with ammonia solution adjust pH value to 10-11, with three
Chloromethanes 20mL extracts 1 time, and chloroform liquid is evaporated, and residue adds methanol 2mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: take berberine hydrochloride reference substance, adds methanol and makes every 1mL solution containing 0.5mg, make
For reference substance solution；
3) point sample, expansion: draw above two solution each 1-2 μ L, put respectively on same silica gel g thin-layer plate, with positive fourth
Alcohol: glacial acetic acid: water=7:1:2 is developing solvent, launches, and takes out, dries, put and inspect under 365nm uviol lamp；In test sample chromatograph,
On position corresponding with reference substance chromatograph, the fluorescence speckle of aobvious same color.
4) result: Fig. 6 is the thin-layer chromatogram of Cortex Phellodendri in Longbi Xintong granule, and result shows: the sample containing Cortex Phellodendri exists
On the position corresponding with berberine hydrochloride reference substance chromatograph, the speckle of aobvious same color.Chromatographic isolation is good, the feature speckle of Cortex Phellodendri
Point is prominent, and the TLC method of foundation can be as the quality determining method of Cortex Phellodendri in Longbi Xintong granule.
Radix Sophorae Flavescentis indentification by TLC in embodiment 7 Longbi Xintong granule
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking,
Filtering, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with dilute hydrochloric acid adjust pH value to 1-2, with ether extraction 2 times, each 20mL,
Water liquid after extraction with ethyl acetate extract 3 times, each 20mL, the water liquid after extraction with ammonia solution adjust pH value to 10-11, with three
Chloromethanes 20mL extracts 1 time, and chloroform liquid is evaporated, and residue adds methanol 2mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: take matrine reference substance, adds methanol and makes every 1mL solution containing 0.2mg, as right
According to product solution；
3) point sample, expansion: draw need testing solution 2-5 μ L, reference substance solution 5 μ L, put respectively in same silica gel G thin layer
On plate, with chloroform: methanol: lower floor's solution of ammonia=5:0.6:0.2, as developing solvent, launches, and takes out, dries, and spray is to change
Good bismuth potassium iodide test solution；In test sample chromatograph, on position corresponding with reference substance chromatograph, the speckle of aobvious same color.
4) result: Fig. 7 is the thin-layer chromatogram of Radix Sophorae Flavescentis in Longbi Xintong granule, and result shows: the sample containing Radix Sophorae Flavescentis exists
On the position that matrine reference substance chromatograph is corresponding, the speckle of aobvious same color.Chromatographic isolation is good, and the characteristic spots of Radix Sophorae Flavescentis is dashed forward
Going out, the TLC method of foundation can be as the quality determining method of Radix Sophorae Flavescentis in Longbi Xintong granule.
Rhizoma Dioscoreae Hypoglaucae indentification by TLC in embodiment 8 Longbi Xintong granule
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking,
Filtering, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with dilute hydrochloric acid adjust pH value to 1-2, with ether extraction 2 times, each 20mL,
Water liquid after extraction with ethyl acetate extract 3 times, each 20mL, the water liquid after extraction with ammonia solution adjust pH value to 10-11, with three
Chloromethanes 20mL extracts 1 time, takes the water liquid 10mL after extraction, extracts 2 times by ethyl acetate, each 10mL, discards ethyl acetate
Liquid, water liquid water saturated n-butanol extraction 2 times, each 10mL, merge n-butanol layer, wash 1 time with ammonia solution 20mL, discard
Ammonia layer, n-butanol layer water bath method, residue adds methanol 2mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: taking Rhizoma Dioscoreae Hypoglaucae control medicinal material 0.5g, add water 30mL, boils 10 minutes, lets cool, filter
Crossing, the saturated n-butanol extraction of filtrate water 2 times, each 20mL, merge n-butyl alcohol liquid, be evaporated, residue adds methanol 2mL to be made molten
Solve, as control medicinal material solution；
3) point sample, expansion: draw each 5 μ L of above two solution, put respectively on same silica gel g thin-layer plate, with three chloromethanes
Alkane: methanol: lower floor's solution of water=13:7:2 is developing solvent, launches, and takes out, dries, and sprays with 10% ethanol solution of sulfuric acid,
105 DEG C are heated to spot development clearly, put respectively and inspect under daylight and 365nm ultra-violet lamp；In test sample chromatograph, with compare
On the corresponding position of medical material chromatograph, the speckle of aobvious same color or fluorescence speckle.
4) result: in Fig. 8, Fig. 9 respectively Longbi Xintong granule, Rhizoma Dioscoreae Hypoglaucae is put and inspected thin-layer chromatogram under daylight and put
Inspecting thin-layer chromatogram under 365nm ultra-violet lamp, result shows: the sample containing Rhizoma Dioscoreae Hypoglaucae with Rhizoma Dioscoreae Hypoglaucae control medicinal material chromatograph
On corresponding position, the speckle of aobvious same color.Chromatographic isolation is good, and the characteristic spots of Rhizoma Dioscoreae Hypoglaucae highlights, the TLC method of foundation
Can be as the quality determining method of Rhizoma Dioscoreae Hypoglaucae in Longbi Xintong granule.
Radix Cyathulae indentification by TLC in embodiment 9 Longbi Xintong granule
1) preparation of need testing solution: take this product 16g, finely ground, add methanol 50mL supersound process 30 minutes, time add shaking,
Filtering, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with ether extraction twice, each 30mL, discards ether solution, and water liquid is with dilute
Hydrochloric acid adjusts pH value to 1-2, extracts twice by ethyl acetate, and each 20mL, acetic acid ethyl fluid is evaporated, residue methanol: acetic acid second
Ester=1:1 dissolves in right amount, is added on 100-200 mesh neutral alumina column, with methanol: the solution 40mL of ethyl acetate=1:1 washes
De-, to collect eluent, be evaporated, residue adds methanol 1mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: separately take Radix Cyathulae control medicinal material 2g, add methanol 50mL, is heated to reflux 1 hour, filter
Crossing, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with ether extraction twice, each 30mL, discards ether solution, the dilute salt of water liquid
Acid adjusts pH value to 1-2, extracts twice by ethyl acetate, and each 20mL, acetic acid ethyl fluid is evaporated, residue methanol: ethyl acetate
=1:1 dissolves in right amount, is added on 100-200 mesh neutral alumina column, with methanol: the solution 40mL eluting of ethyl acetate=1:1,
Collecting eluent, be evaporated, residue adds methanol 1mL makes dissolving, as reference substance solution；
3) point sample, expansion: draw each 5 μ L of above two solution, put respectively on same silica gel g thin-layer plate, with three chloromethanes
Alkane: methanol: formic acid=10:1.5:2 is developing solvent, launches, and takes out, dries, and spray, with 10% ethanol solution of sulfuric acid, adds at 105 DEG C
Heat is clear to spot development, puts and inspects under 365nm ultra-violet lamp；In test sample chromatograph, in position corresponding with control medicinal material chromatograph
Put, the fluorescence speckle of aobvious same color.
4) result: Figure 10 is the thin-layer chromatogram of Radix Cyathulae in Longbi Xintong granule, and result shows: containing the sample of Radix Cyathulae
Product, on the position corresponding with Radix Cyathulae control medicinal material chromatograph, show the speckle of same color.Chromatographic isolation is good, the spy of Radix Cyathulae
Levying speckle to highlight, the TLC method of foundation can be as the quality determining method of Radix Cyathulae in Longbi Xintong granule.
The berberine hydrochloride of Cortex Phellodendri in embodiment 10 high effective liquid chromatography for measuring Longbi Xintong granule
1) preparation of need testing solution: take Longbi Xintong granule appropriate, finely ground, take 2g, accurately weighed, put in conical flask,
The accurate hydrochloric acid that adds: the solution 50mL of methanol=1:100, weighed weight, supersound process 30 minutes, lets cool, weighed weight, uses salt
Acid: the solution of methanol=1:100 supplies less loss weight, shakes up, filters, accurate absorption subsequent filtrate 3mL, is added in 100-200 mesh
Property alumina column on, with methanol 50mL eluting, collect eluent, water bath method, residue dissolves with methanol and constant volume holds in 10mL
In measuring bottle, shake up, filter, to obtain final product；
2) preparation of reference substance solution: weigh berberine hydrochloride reference substance appropriate, adds methanol and makes every 1mL containing 0.03mg's
Solution, shakes up, and to obtain final product；
3) HPLC chromatogram condition: octadecylsilane chemically bonded silica is filler；Acetonitrile: 0.05mol/L sodium dihydrogen phosphate
Solution=30:70 is flowing phase；Detection wavelength is 346nm, and number of theoretical plate is calculated by berberine hydrochloride peak should be not less than 4000；
4) assay method: precision draws reference substance solution and need testing solution 10uL respectively, injects high performance liquid chromatograph,
According to step 3) described chromatographic condition mensuration, to obtain final product.
The amygdaloside of Semen Persicae in embodiment 11 high effective liquid chromatography for measuring Longbi Xintong granule
1) preparation of need testing solution: take Longbi Xintong granule appropriate, finely ground, take 5g, accurately weighed, put tool plug conical flask
In, accurate addition water 50mL, weighed weight, supersound process 30 minutes, let cool, more weighed weight, the weight of less loss is supplied with water,
Shaking up, centrifugal, accurate Aspirate supernatant 10mL puts in separatory funnel, extracts 2 times by ethyl acetate, each 20mL, discards acetic acid
Ethyl ester liquid, water liquid is put in evaporating dish, is washed with a small amount separatory funnel, is incorporated in the lump in evaporating dish, and water-bath is waved to without acetic acid
Ethyl ester taste, lets cool, and is transferred in 25mL measuring bottle, constant volume, shakes up, to obtain final product；
2) preparation of reference substance solution: weigh amygdaloside reference substance appropriate, adds methanol and makes molten containing 0.1mg of every 1mL
Liquid, shakes up, and to obtain final product；
3) HPLC chromatogram condition: octadecylsilane chemically bonded silica is filler；Methanol: water=18:82 is flowing phase；Inspection
Survey wavelength is 210nm, and number of theoretical plate is calculated by amygdaloside should be not less than 4000；
4) assay method: precision draws reference substance solution and need testing solution 5uL respectively, injects high performance liquid chromatograph,
According to step 3) described chromatographic condition mensuration, to obtain final product.
Above example is only the preferred embodiment of the present invention, therefore it describes more concrete and detailed, but can not be
And it is interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that, for the person of ordinary skill of the art, without departing from
On the premise of the principle of the invention and design, it is also possible to making some modifications and improvement, these broadly fall into protection scope of the present invention.
Claims (11)
1. the quality determining method of the Chinese patent medicine Longbi Xintong granule treating chronic prostatitis, it is characterised in that the party
Method differentiates Cortex Cinnamomi, Herba Violae, Radix Scrophulariae, Cortex Phellodendri, Radix Sophorae Flavescentis, Rhizoma Dioscoreae Hypoglaucae and Radix Cyathulae in preparation with thin layer chromatography, uses simultaneously
HPLC measures the amygdaloside content in the content of berberine hydrochloride of Cortex Phellodendri in preparation and Semen Persicae.
Method the most according to claim 1, it is characterised in that thin layer chromatography differentiates that in preparation, Cortex Cinnamomi includes following step
Rapid:
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking, filter
Crossing, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, adjusts pH value to 1-2 with dilute hydrochloric acid, with ether extraction 2 times, each 20mL, closes
And ether solution, low temperature volatilizes, and residue adds methanol 2mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: take cinnamic aldehyde reference substance and add ethanol and make every 1mL containing the solution of 1 μ L, molten as reference substance
Liquid；
3) point sample, expansion: draw need testing solution 2-5 μ L, reference substance solution 2 μ L, put respectively on same silica gel g thin-layer plate,
With 60-90 DEG C of petroleum ether: ethyl acetate=17:3, as developing solvent, launches, and takes out, dries, and spray is with dinitrophenylhydrazine ethanol test solution；
In test sample chromatograph, on position corresponding with reference substance chromatograph, the speckle of aobvious same color.
Method the most according to claim 1, it is characterised in that thin layer chromatography differentiates that in preparation, Herba Violae includes following
Step:
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking, filter
Crossing, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, adjusts pH value to 1-2 with dilute hydrochloric acid, with ether extraction 2 times, each 20mL, closes
And ether solution, low temperature volatilizes, and residue adds methanol 2mL makes dissolving, as need testing solution；
2) preparation of control medicinal material solution: separately take Herba Violae control medicinal material 2g, adds methanol 20mL, supersound extraction 20 minutes, filter
Crossing, be evaporated, residue adds hot water 20mL makes dissolving, filters, with dilute hydrochloric acid adjust pH value to 1-2, with ether extraction 2 times, each 20mL,
Merging ether solution, volatilize, residue adds methanol 2mL makes dissolving, as control medicinal material solution；
3) point sample, expansion: draw above two solution each 2-5 μ L, put respectively on same silica gel g thin-layer plate, with chloroform:
Acetone: formic acid=25:20:4 is developing solvent, launches, and takes out, dries, put and inspect under 365nm ultra-violet lamp；In test sample chromatograph,
On position corresponding with control medicinal material chromatograph, the fluorescence principal spot of aobvious same color.
Method the most according to claim 1, it is characterised in that thin layer chromatography differentiates that in preparation, Radix Scrophulariae includes following step
Rapid:
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking, filter
Crossing, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, adjusts pH value to 1-2 with dilute hydrochloric acid, with ether extraction 2 times, each 20mL, carries
Water liquid ethyl acetate after taking is extracted 3 times, each 20mL, and combined ethyl acetate solution is evaporated, and residue is with a small amount of 50% methanol
Dissolving, be added on 100-200 mesh neutral alumina column, with 50% methanol 30mL eluting, eluent is evaporated, and residue adds methanol
0.5mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: take Radix Scrophulariae control medicinal material 1g, adds methanol 20mL, supersound extraction 30 minutes, filters, be evaporated,
The residue 20mL that adds water makes dissolving, extracts 2 times by ethyl acetate, each 20mL, combined ethyl acetate liquid, is evaporated, and residue adds methanol
1mL makes dissolving, as control medicinal material solution；
3) point sample, expansion: draw need testing solution 5-10 μ L, control medicinal material solution 4 μ L, put respectively in same silica gel g thin-layer plate
On, with chloroform: methanol: water: lower floor's solution of formic acid=12:4:1:1, as developing solvent, launches, and takes out, dries, and spray is with 5%
Vanillin-sulfuric acid solution, 105 DEG C to be heated to spot development clear；In test sample chromatograph, in position corresponding with control medicinal material chromatograph
Put, the speckle of aobvious same color.
Method the most according to claim 1, it is characterised in that thin layer chromatography differentiates that in preparation, Cortex Phellodendri includes following step
Rapid:
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking, filter
Crossing, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, adjusts pH value to 1-2 with dilute hydrochloric acid, with ether extraction 2 times, each 20mL, carries
Water liquid ethyl acetate after taking is extracted 3 times, each 20mL, and the ammonia solution of the water liquid after extraction adjusts pH value to 10-11, uses trichlorine
Methane 20mL extracts 1 time, and chloroform liquid is evaporated, and residue adds methanol 2mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: take berberine hydrochloride reference substance, adds methanol and makes every 1mL solution containing 0.5mg, as right
According to product solution；
3) point sample, expansion: draw above two solution each 1-2 μ L, put respectively on same silica gel g thin-layer plate, with n-butyl alcohol: ice
Acetic acid: water=7:1:2 is developing solvent, launches, and takes out, dries, put and inspect under 365nm uviol lamp；In test sample chromatograph, with right
According on the corresponding position of product chromatograph, show the fluorescence speckle of same color.
Method the most according to claim 1, it is characterised in that thin layer chromatography differentiates that in preparation, Radix Sophorae Flavescentis includes following step
Rapid:
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking, filter
Crossing, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, adjusts pH value to 1-2 with dilute hydrochloric acid, with ether extraction 2 times, each 20mL, carries
Water liquid ethyl acetate after taking is extracted 3 times, each 20mL, and the ammonia solution of the water liquid after extraction adjusts pH value to 10-11, uses trichlorine
Methane 20mL extracts 1 time, and chloroform liquid is evaporated, and residue adds methanol 2mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: take matrine reference substance, adds methanol and makes every 1mL solution containing 0.2mg, as reference substance
Solution；
3) point sample, expansion: draw need testing solution 2-5 μ L, reference substance solution 5 μ L, put respectively in same silica gel g thin-layer plate
On, with chloroform: methanol: lower floor's solution of ammonia=5:0.6:0.2, as developing solvent, launches, and takes out, dries, and sprays with improvement
Bismuth potassium iodide test solution；In test sample chromatograph, on position corresponding with reference substance chromatograph, the speckle of aobvious same color.
Method the most according to claim 1, it is characterised in that thin layer chromatography differentiates that in preparation, Rhizoma Dioscoreae Hypoglaucae includes following step
Rapid:
1) preparation of need testing solution: take this product 10g, finely ground, add methanol 50mL, supersound extraction 30 minutes, time add shaking, filter
Crossing, filtrate is evaporated, and the residue 20mL that adds water makes dissolving, adjusts pH value to 1-2 with dilute hydrochloric acid, with ether extraction 2 times, each 20mL, carries
Water liquid ethyl acetate after taking is extracted 3 times, each 20mL, and the ammonia solution of the water liquid after extraction adjusts pH value to 10-11, uses trichlorine
Methane 20mL extracts 1 time, takes the water liquid 10mL after extraction, extracts 2 times by ethyl acetate, each 10mL, discards acetic acid ethyl fluid,
Water liquid water saturated n-butanol extraction 2 times, each 10mL, merge n-butanol layer, wash 1 time with ammonia solution 20mL, discard ammonia
Water layer, n-butanol layer water bath method, residue adds methanol 2mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: taking Rhizoma Dioscoreae Hypoglaucae control medicinal material 0.5g, add water 30mL, boils 10 minutes, lets cool, and filters, filter
Liquid water saturated n-butanol extraction 2 times, each 20mL, merge n-butyl alcohol liquid, be evaporated, residue adds methanol 2mL makes dissolving, as
Control medicinal material solution；
3) point sample, expansion: draw each 5 μ L of above two solution, put respectively on same silica gel g thin-layer plate, with chloroform: first
Alcohol: lower floor's solution of water=13:7:2 is developing solvent, launches, and takes out, dries, and spray, with 10% ethanol solution of sulfuric acid, adds at 105 DEG C
Heat is clear to spot development, puts respectively and inspects under daylight and 365nm ultra-violet lamp；In test sample chromatograph, with control medicinal material color
Compose on corresponding position, the speckle of aobvious same color or fluorescence speckle.
Method the most according to claim 1, it is characterised in that thin layer chromatography differentiates that in preparation, Radix Cyathulae includes following step
Rapid:
1) preparation of need testing solution: take this product 16g, finely ground, add methanol 50mL supersound process 30 minutes, time add shaking, filter,
Filtrate is evaporated, and the residue 20mL that adds water makes dissolving, with ether extraction twice, each 30mL, discards ether solution, and water liquid dilute hydrochloric acid is adjusted
PH value, to 1-2, extracts twice by ethyl acetate, and each 20mL, acetic acid ethyl fluid is evaporated, residue methanol: ethyl acetate=1:1
Appropriate dissolve, be added on 100-200 mesh neutral alumina column, with methanol: the solution 40mL eluting of ethyl acetate=1:1, collect
Eluent, is evaporated, and residue adds methanol 1mL makes dissolving, as need testing solution；
2) preparation of reference substance solution: separately take Radix Cyathulae control medicinal material 2g, add methanol 50mL, is heated to reflux 1 hour, filters, filter
Liquid is evaporated, and the residue 20mL that adds water makes dissolving, with ether extraction twice, each 30mL, discards ether solution, and water liquid dilute hydrochloric acid adjusts pH
Being worth to 1-2, extract twice by ethyl acetate, each 20mL, acetic acid ethyl fluid is evaporated, residue methanol: ethyl acetate=1:1 fits
Amount is dissolved, and is added on 100-200 mesh neutral alumina column, with methanol: the solution 40mL eluting of ethyl acetate=1:1, collection is washed
De-liquid, is evaporated, and residue adds methanol 1mL makes dissolving, as reference substance solution；
3) point sample, expansion: draw each 5 μ L of above two solution, put respectively on same silica gel g thin-layer plate, with chloroform: first
Alcohol: formic acid=10:1.5:2 is developing solvent, launches, and takes out, dries, and spray, with 10% ethanol solution of sulfuric acid, is heated to speckle at 105 DEG C
Point colour developing is clear, puts and inspects under 365nm ultra-violet lamp；In test sample chromatograph, on position corresponding with control medicinal material chromatograph, aobvious
The fluorescence speckle of same color.
9. according to the method according to any one of claim 1-8, it is characterised in that in Preparations by HPLC
The content of berberine of Cortex Phellodendri comprises the following steps:
1) preparation of need testing solution: take Longbi Xintong granule appropriate, finely ground, take 2g, accurately weighed, put in conical flask, accurate
Add hydrochloric acid: the solution 50mL of methanol=1:100, weighed weight, supersound process 30 minutes, let cool, weighed weight, with hydrochloric acid:
Methanol=1:100 solution supplies less loss weight, shakes up, and filters, accurate absorption subsequent filtrate 3mL, is added in 100-200 mesh neutral alumina
On aluminum post, with methanol 50mL eluting, collecting eluent, water bath method, residue dissolves with methanol and constant volume is in 10mL volumetric flask,
Shake up, filter, to obtain final product；
2) preparation of reference substance solution: weigh berberine hydrochloride reference substance appropriate, adds methanol and makes molten containing 0.03mg of every 1mL
Liquid, shakes up, and to obtain final product；
3) HPLC chromatogram condition: octadecylsilane chemically bonded silica is filler；Acetonitrile: 0.05mol/L sodium dihydrogen phosphate
=30:70 is flowing phase；Detection wavelength is 346nm, and number of theoretical plate is calculated by berberine hydrochloride peak should be not less than 4000；
4) assay method: precision draws reference substance solution and need testing solution 10uL respectively, injects high performance liquid chromatograph, according to
Step 3) described chromatographic condition mensuration, to obtain final product.
Method the most according to claim 9, it is characterised in that the bitter Fructus Pruni of Semen Persicae in Preparations by HPLC
Core glycosides content comprises the following steps:
1) preparation of need testing solution: take Longbi Xintong granule appropriate, finely ground, take 5g, accurately weighed, put in tool plug conical flask,
Accurate addition water 50mL, weighed weight, supersound process 30 minutes, let cool, more weighed weight, supply the weight of less loss with water, shake
Even, centrifugal, accurate Aspirate supernatant 10mL puts in separatory funnel, extracts 2 times by ethyl acetate, each 20mL, discards acetic acid second
Ester liquid, water liquid is put in evaporating dish, is washed with a small amount separatory funnel, is incorporated in the lump in evaporating dish, and water-bath is waved to without acetic acid second
Ester taste, lets cool, and is transferred in 25mL measuring bottle, constant volume, shakes up, to obtain final product；
2) preparation of reference substance solution: weigh amygdaloside reference substance appropriate, adds methanol and makes every 1mL solution containing 0.1mg, shake
Even, to obtain final product；
3) HPLC chromatogram condition: octadecylsilane chemically bonded silica is filler；Methanol: water=18:82 is flowing phase；Detection ripple
A length of 210nm, number of theoretical plate is calculated by amygdaloside should be not less than 4000；
4) assay method: precision draws reference substance solution and need testing solution 5uL respectively, injects high performance liquid chromatograph, according to
Step 3) described chromatographic condition mensuration, to obtain final product.
11. according to the method according to any one of claim 1-8, it is characterised in that in Preparations by HPLC
The amygdaloside content of Semen Persicae comprises the following steps:
1) preparation of need testing solution: take Longbi Xintong granule appropriate, finely ground, take 5g, accurately weighed, put in tool plug conical flask,
Accurate addition water 50mL, weighed weight, supersound process 30 minutes, let cool, more weighed weight, supply the weight of less loss with water, shake
Even, centrifugal, accurate Aspirate supernatant 10mL puts in separatory funnel, extracts 2 times by ethyl acetate, each 20mL, discards acetic acid second
Ester liquid, water liquid is put in evaporating dish, is washed with a small amount separatory funnel, is incorporated in the lump in evaporating dish, and water-bath is waved to without acetic acid second
Ester taste, lets cool, and is transferred in 25mL measuring bottle, constant volume, shakes up, to obtain final product；
2) preparation of reference substance solution: weigh amygdaloside reference substance appropriate, adds methanol and makes every 1mL solution containing 0.1mg, shake
Even, to obtain final product；
3) HPLC chromatogram condition: octadecylsilane chemically bonded silica is filler；Methanol: water=18:82 is flowing phase；Detection ripple
A length of 210nm, number of theoretical plate is calculated by amygdaloside should be not less than 4000；
4) assay method: precision draws reference substance solution and need testing solution 5uL respectively, injects high performance liquid chromatograph, according to
Step 3) described chromatographic condition mensuration, to obtain final product.
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| CN110736803A (en) * | 2019-11-20 | 2020-01-31 | 贵州缔谊健康制药有限公司 | method for detecting Qibai burn ointment |
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| CN115754094A (en) * | 2022-07-19 | 2023-03-07 | 石家庄科迪药业有限公司 | Fingerprint of urine retention relieving preparation and establishing method and application thereof |
| CN115754094B (en) * | 2022-07-19 | 2024-04-19 | 石家庄科迪药业有限公司 | Finger print of uroschesis relieving preparation and establishing method and application thereof |
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