CN110376317A - A kind of Cortex Phellodendri reference extract and its preparation method and application - Google Patents
A kind of Cortex Phellodendri reference extract and its preparation method and application Download PDFInfo
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- CN110376317A CN110376317A CN201810330280.0A CN201810330280A CN110376317A CN 110376317 A CN110376317 A CN 110376317A CN 201810330280 A CN201810330280 A CN 201810330280A CN 110376317 A CN110376317 A CN 110376317A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
Abstract
The invention discloses a kind of Cortex Phellodendri reference extracts and its preparation method and application, the extract is yellow dried powder, it is made of phellodendron extract containing weight percent 67-84% and auxiliary material 16-33%, is wherein not less than 30% containing content of berberine in phellodendron extract；Cortex Phellodendri reference extract consistency is good, character is stable, uniform for this, can be used for the quality control of Cortex Phellodendri medicinal material, Cortex Phellodendri preparation, measures to the analysis of active compound qualitative, quantitative.
Description
Technical field
The invention belongs to chemical fields, more particularly, to a kind of Cortex Phellodendri reference extract and preparation method thereof.
Background technique
Drug inspection is an important means for verifying drug quality, more and more with being widely used for instrument analysis
Ground uses pharmaceutical standard material.Special cross-reference entry object of the Chang Huiyong reference substance as inspection drug quality in drug inspection work,
Using it as the reference standard for determining authenticity of medicament superiority and inferiority.Reference substance has highly important status in drug inspection.
In present Chinese medicine standard, the mode of single component control quality is difficult to reflect traditional Chinese medicine quality well, and use is a variety of
The method of ingredient or Characteristic chromatographic peak comprehensively control quality increasingly attracts people's attention.However, the reference substance of commercialization
Supply falls short of demand and high testing cost limits multicomponent quality control answering in actual production, scientific research and supervision area
With.It not only can solve big, the in short supply problem of monomer reference substance preparation difficulty using reference extract, but also can achieve to index
The purpose of Composition Control has important meaning to the quality control and standard execution of Chinese medicine.
Existing traditional Chinese medicine quality control mode is substantially the development along Natural Medicine Chemistry, a certain or several to Chinese medicine
Effective component is the analysis method and the not only design of difinite quality but also the quality standard that can be quantified of target, referring to external botanical medicine
Method of quality control uses for reference the mode of chemicals quality control, and establishes corresponding simple physical and chemical mirror by document report
Not, quality standard of the developing deeply to identification and assay based on spectrum, chromatography.It is all multicomponent per Chinese medicine simply
Synthesis, which dictates that its distinctive globality and ambiguity, also indicate that one or two using in medicinal material even several ingredients as
There are significant limitations for the evaluation method of quality of medicinal material.
1990 editions " Chinese Pharmacopoeias " increase the indentification by TLC of control medicinal material, so that the identification of Chinese medicine and Chinese patent drug has
Very big progress.Chinese medicine and external herbal medicine increasingly pay attention to multicomponent or multi-component detection, such as German ginkgo at present
The quality of leaf extract controls.The analysis method of finger-print can carry out quality control to medicinal material on the whole, now still
So there is very big researching value.At present Chinese Pharmacopoeia in terms of controlling quality of medicinal material there are two types of " reference substance ", chemical reference substance and
Chinese medicine control medicinal material.Wherein chemical reference substance can be used for Qualitive test and the quantitative analysis of medicinal material, and Chinese medicine control medicinal material is then used for
Microscopical characters and thin layer identify.However, chemical reference substance and Chinese medicine control medicinal material have its limitation in traditional Chinese medicine quality control
Property.Firstly, chemical component diversification, single or several compounds can not reflect the overall picture of medicinal material in Chinese medicine, and it is existing
The phenomenon that standard often has many loopholes, adulterates happens occasionally.Chinese medicine control medicinal material is by the place of production, the shadow of growing environment
It rings, it is difficult to ensure that per batch of uniform quality, and it is only used for Qualitive test, it can not reflect the height of medicinal ingredient content.
There are four basic demand (ASCS) for Chinese medicine reference extract: Authenticity, crude drug source is reliable, has and represents
Property；Specificity, the detection method used have specificity；Con-sistency, different batches reference extract should be kept
Unanimously；Stability, character are stable, uniform, easy to use.Refer to TLC Fingerprints and high performance liquid chromatography
The method of line map, Chinese medicine reference extract are able to satisfy the needs of Qualitive test.It can also be by the reference extract of markization
One step carries out the even quantitative analysis of sxemiquantitative.Chinese medicine reference extract will have important justice to traditional Chinese medicine quality control.
Cortex Phellodendri medicinal material (for rutaceae wampee Phellodendron chinense Schneid, dry tree
Skin).In the Chinese Pharmacopoeia quality standard of Chinese Pharmacopoeia Commission's publication, thin-layered chromatography is examined when to Cortex Phellodendri as control medicinal material
Phellodendrine, the content of high effective liquid chromatography for measuring jamaicin and phellodendrine are surveyed, Cortex Phellodendri control medicinal material operates ratio as test sample
It is more complex, and medicinal material itself is unstable.During drug routine check, effective substance is unstable, and product quality cannot get
Effectively control.Since Chinese medicine reference extract has an above-mentioned specific requirement, currently available technology does not have also or does not have mature Huang
Cypress reference extract and preparation method thereof replaces the application of Cortex Phellodendri control medicinal material.
Summary of the invention
In order to solve the above-mentioned technical problem, the purpose of the present invention is to provide one kind in order to solve the above-mentioned technical problem, this
Invention is not comprehensive to examination criteria present in Cortex Phellodendri preparation or the detection of Cortex Phellodendri medicinal material, high using a variety of reference substance testing costs
The problem of, the present invention provides a kind of pair of Cortex Phellodendri medicinal material, phellodendron extract and its compound in Cortex Phellodendri preparation while detecting
Cortex Phellodendri reference extract, Cortex Phellodendri reference extract consistency is good, character is stable, uniform for this, can be used for Cortex Phellodendri medicinal material, Cortex Phellodendri
The quality of prescription controls, and measures to the analysis of active compound qualitative, quantitative.
The present invention also provides the preparation methods of Cortex Phellodendri reference extract.This method is easy to operate, at low cost, reproducible
And recovery rate is high.
The present invention also provides the controls for the sample that control medicinal material detection ingredient containing Cortex Phellodendri is replaced with Cortex Phellodendri reference extract
Method.
The present invention is achieved through the following technical solutions:
A kind of Cortex Phellodendri reference extract of the invention, the extract are yellow dried powder, are by containing weight percent
It is formed than phellodendron extract 67-84% and auxiliary material 16-33%, is wherein not less than 30% containing jamaicin in phellodendron extract.
Further preferably, a kind of Cortex Phellodendri reference extract of the invention, content >=34% of Berberine.
The jamaicin in chromatogram that Cortex Phellodendri reference extract of the present invention uses thin-layered chromatography to detect
The chromatographic peak of fluorescence spot and the chromatogram detected using high performance liquid chromatography at jamaicin position should respectively with its
Corresponding chemical reference substance or its corresponding raw medicinal material are consistent.
What Cortex Phellodendri reference extract of the present invention was prepared by the following method:
(1) Cortex Phellodendri dry cream: the Cortex Phellodendri medicinal powder methanol ultrasonic extraction of separate sources and batch is taken, filtrate 1 is filtered to obtain；
The dregs of a decoction again with methanol ultrasonic extraction 1-3 times, 1-3 filtrate of collection mixes with filtrate 1, is evaporated to obtain Cortex Phellodendri dry cream；
(2) phellodendron extract: Cortex Phellodendri dry cream is dissolved in methanol, add auxiliary material be evaporated be ground up, sieved up to Cortex Phellodendri extract
Object；
(3) Cortex Phellodendri reference extract: separate sources or the phellodendron extract of batch are subjected to allotment and blent, Cortex Phellodendri pair is obtained
According to extract.
In above-mentioned steps 1 w/v of Cortex Phellodendri medicinal powder and methanol be 1:8~1:50 (preferably 1:8~1:20, it is excellent
Select 1:10)；The ultrasonic time is 30~60min, and power is that (preferably ultrasonic time is 30min to 100W~3kW, and power is
500W)。
Filtering described in step 1 is with Medium speed filter paper or 2000 mesh net filtrations.
Methanol usage is 2-5 times of dry cream weight in the step 2.
10%~60% (preferably 20-40%) of dry cream weight is added in the step 2 in Cortex Phellodendri dry cream methanol solution
Auxiliary material；
The auxiliary material includes but is not limited to: the such as superfine silica gel powder, medical starch
It is preferred that auxiliary material is 20-50% (preferably 40%) superfine silica gel powder and/or 10-30% (preferably 20%) medical starch.Institute
Stating auxiliary material and dosing can guarantee that extract character is stable, uniform and easy to use.
Being ground up, sieved after being evaporated described in step 2 was 90~200 mesh (preferably 200 mesh) the screen to filtrate.
Allotment described in step 3 refers specifically to: the phellodendron extract of separate sources or batch being deployed, should be made final
Cortex Phellodendri reference extract in correspond to crude drug ratio be 1:(2-5) (g/g), (preferably 1:4g/g), wherein in phellodendron extract
It is not less than 30% containing jamaicin.
Further preferably, a kind of Cortex Phellodendri reference extract of the invention, content >=34% of Berberine.
The present invention further includes with thin-layered chromatography and/or high performance liquid chromatography between the step 2 and step 3 to not
The step of being detected with the jamaicin of the phellodendron extract of batch.
For reliability, content consistency, the homogeneity for guaranteeing Cortex Phellodendri reference extract of the invention.It is preferred that in step (1)
The preceding screening step for increasing different crude drug sources: by separate sources or the raw medicinal material of batch with thin-layered chromatography and/or efficiently
Liquid chromatography is detected；Then thin-layered chromatography and/or efficient liquid are passed through to the raw medicinal material of separate sources or batch
The content of chromatogram and/or measurement that phase chromatography obtains is compared analysis, and the content for rejecting chromatogram and/or measurement is different
Normal raw medicinal material retains the system of the Cortex Phellodendri dry cream of the consistent raw medicinal material progress next step of content of chromatogram and/or measurement
It is standby.
Cortex Phellodendri reference extract of the invention exists in a unit, and the unit dosage form refers to the list of preparation
Position, such as every of tablet, every of capsule,
Preparation of the invention includes but is not limited to tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard
Capsule, soft capsule, oral solution, mouth containing agent, granule, electuary, pill, powder, paste, sublimed preparation, suspension, pulvis,
Solution, injection, suppository, ointment, emplastrum, creme, spray, drops, patch.
Pharmaceutical preparation of the invention, the preparation of oral administration can contain common excipient, such as adhesive, filling
Agent, diluent, tablet agent, lubricant, disintegrating agent, colorant, flavoring agent and wetting agent when necessary can be coated tablet.
Applicable filler includes cellulose, mannitol, lactose and other similar fillers.Suitable disintegrating agent include starch,
Polyvinylpyrrolidone and starch derivatives, such as sodium starch glycollate.Suitable lubricant includes, such as magnesium stearate.
Suitable pharmaceutically acceptable wetting agent includes lauryl sodium sulfate.Mixing, filling, the common method such as tabletting can be passed through
Prepare solid oral composition.Carrying out mixing repeatedly can be such that active material is distributed in entirely using those of a large amount of fillers group
It closes in object.
The form of oral liquid for example can be aqueous or oily suspensions, solution, emulsion, syrup or elixir,
Or it can be a kind of dry products that can be compounded with water or other suitable carriers before use.This liquid preparation can contain
There are conventional additive, such as suspending agent, such as sorbierite, syrup, methylcellulose, gelatin, hydroxyethyl cellulose, carboxylic first
Base cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifier, such as lecithin, anhydro sorbitol monooleate or Ah
Draw primary glue；Non-aqueous carrier (they may include edible oil), for example, apricot kernel oil, fractionated coconut oil, such as glycerol ester oil
Property ester, propylene glycol or ethyl alcohol；Preservative, such as para hydroxybenzene methyl esters or propylparaben or sorbic acid, and if
It needs, contains conventional flavouring agent or colorant.
For injection, the fluid unit dosage form of preparation contains active material and sterile carrier of the invention.According to carrier
And concentration, this compound can be suspended or be dissolved.The preparation of solution is usually by the way that active material is dissolved in a kind of load
In body, disinfection is filtered before being loaded into a kind of suitable bottle or ampoule, is then sealed.For example a kind of local anaesthesia of auxiliary material
Agent, preservative and buffer are also soluble in this carrier.In order to improve its stability, can be incited somebody to action after being packed into bottle
This composition frost, and under vacuum remove water.
Cortex Phellodendri reference extract of the invention is optionally added suitable pharmaceutically acceptable when being prepared into medicament
Carrier, the pharmaceutically acceptable carrier are selected from: mannitol, sorbierite, sodium pyrosulfite, sodium hydrogensulfite, thiosulfuric acid
Sodium, cysteine hydrochloride, thioacetic acid, methionine, injection Vitamin B_6 DTA disodium, Ethylenediaminetetraacetic Acid Calcium Salt, the carbonic acid of monovalence alkali metal
Salt, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate,
Xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative,
Cellulose and its derivates, alginates, gelatin, polyvinylpyrrolidone, glycerol, POLYSORBATE 80, agar, calcium carbonate, bicarbonate
It is calcium, surfactant, polyethylene glycol, cyclodextrin, beta-cyclodextrin, phospholipid material, kaolin, talcum powder, calcium stearate, hard
Fatty acid magnesium etc..
Cortex Phellodendri reference extract of the invention is also used as Cortex Phellodendri standard extract, uses or for bulk pharmaceutical chemicals as medicine
The effective component of compositions, weight ratio 0.01-99.9%.
The present invention provides the preparation method of Cortex Phellodendri reference extract, steps are as follows: seeing Fig. 4 process flow chart
1, Cortex Phellodendri dry cream: the Cortex Phellodendri medicinal powder methanol ultrasonic extraction of separate sources and batch is taken, filtrate 1 is filtered to obtain；
The dregs of a decoction again with methanol ultrasonic extraction 1-3 times, 1-3 filtrate of collection mixes with filtrate 1, is evaporated to obtain Cortex Phellodendri dry cream；
2, phellodendron extract: Cortex Phellodendri dry cream is dissolved in methanol, adds auxiliary material and is evaporated and is ground up, sieved up to phellodendron extract；
3, phellodendron extract detects: being extracted with thin-layered chromatography and/or high performance liquid chromatography to the Cortex Phellodendri of different batches
The step of Berberine hydrochloride of object is detected；
4, Cortex Phellodendri reference extract: the phellodendron extract after will test carries out allotment and blends, and obtains Cortex Phellodendri reference extract.
The w/v of Cortex Phellodendri medicinal powder and methanol is 1:8~1:50 (preferably 1:10) in above-mentioned steps 1；It is described super
The sound time is 30~60min, and power is 100W~3kW (preferably ultrasonic time is 30min, power 500W).Institute in step 1
The filtering stated is with Medium speed filter paper or 2000 mesh net filtrations.
Methanol usage is 2-5 times of dry cream weight in the step 2.
Enter 10%~60% auxiliary material of dry cream weight, the auxiliary material in the step 2 in Cortex Phellodendri dry cream methanol solution
Including but not limited to: superfine silica gel powder, medical starch etc..Further preferred auxiliary material is 20-50% (preferably 40%) superfine silica gel powder,
And/or 10-30% (preferably 20%) medical starch.The auxiliary material, which is dosed, can guarantee that extract character is stable, uniformly and is convenient for
It uses.
Being ground up, sieved after being evaporated in the step 2 was 90~200 mesh (preferably 200 mesh) the screen to filtrate.
Detection described in above-mentioned steps 3, wherein the thin-layered chromatography of phellodendron extract includes the following steps:
(1) chemical reference substance solution: precision weighs jamaicin, and the solution of 0.1mg/ml is configured to methanol；
(2) phellodendron extract solution: accurate phellodendron extract is ultrasonically treated with methanol, constant volume, and being made into concentration is 2.5mg/
Ml solution crosses 0.22 μm of filter membrane to obtain the final product；
(3) the thin-layered chromatography testing conditions are as follows:
Lamellae: G60 prefabricated board；
Point sample: 0.5 μ l, the long 10mm of ribbon point sample；
Solvent: butyl acetate: methanol: isopropanol: ammonium hydroxide=5:2:1.5:1.2；
Expansion mode: double flute expansion cylinder, concentrated ammonia liquor test solution pre-equilibrate, solvent usage amount: concentrated ammonia liquor usage amount=1:(1-
2)
The drying of lamellae: the lamellae after point sample is placed in phosphorus pentoxide vacuum desiccator, guarantees the drying of lamellae；
It inspects: being placed under UV366nm and inspect；
It is preferred that solvent expansion mode of the present invention, if adding exhibition using the side double flute expansion cylinder (20cm*10cm)
Agent 10ml is opened, 20ml concentrated ammonia liquor test solution is placed in the other side, and when the specification of the developing tank such as used is 10*10cm, side adds expansion
10ml concentrated ammonia liquor test solution is placed in agent 10ml, the other side.
Wherein the high performance liquid chromatography of phellodendron extract includes the following steps:
(1) preparation of chemical reference substance solution: precision weighs jamaicin, and the solution of 0.1mg/ml is configured to methanol；
(2) preparation of phellodendron extract test solution: taking phellodendron extract that methanol-hydrochloric acid (100:1) is added, at ultrasound
Reason, lets cool, and is configured to the solution that concentration is 0.24mg/ml, and filtration takes subsequent filtrate to get phellodendron extract solution；
(3) chromatographic condition:
The high performance liquid chromatography testing conditions:
Chromatographic apparatus: high performance liquid chromatograph is furnished with DAD detector
Chromatographic column: C18Column (250mm × 4.6mm, 5 μm)；
Mobile phase: A- acetonitrile, the aqueous solution 1:1 of B- -0.3% triethylamine Han 0.3% phosphoric acid；
Gradient elution program: 0-25min:15%A → 25%A,
25-40min:25%A → 28%A,
40-50min:28%A；
Detection wavelength 270nm (DAD detector)；Flow velocity 0.8ml/min；10 μ l of sample volume；25 DEG C of column temperature.
(4) detection method: accurate each 10 μ l of phellodendron extract test solution injects liquid chromatograph, records chromatogram.
Allotment described in above-mentioned steps 4 refers specifically to: the phellodendron extract of separate sources or batch being deployed, should be made most
It is 1:(2-5 that crude drug ratio is corresponded in whole Cortex Phellodendri reference extract), preferably 1:4 (g/g) wherein contains in phellodendron extract
There is content of berberine not less than 30%.
Further preferably, a kind of Cortex Phellodendri reference extract of the invention, content >=34% of Berberine.
The range of the content of each ingredient in allotment mark of the present invention, which is also that experiment is comprehensive repeatedly by repeatedly, protects
The optimum data that the qualitative, consistency and subsequent application etc. of keeping steady obtain.
The present invention also provides the discrimination method or Cortex Phellodendri of a kind of Cortex Phellodendri medicinal material or Chinese materia medica preparation or effective components containing Cortex Phellodendri
Medicinal material or Chinese materia medica preparation method of quality control, by the above-mentioned Cortex Phellodendri being prepared according to extract, sample to be tested is with thin
Layer chromatography and/or high performance liquid chromatography are detected, and comparison differentiates.
Beneficial effects of the present invention are illustrated by following test examples
The preparation method of Cortex Phellodendri reference extract of the invention use slightly mention, refine, plus auxiliary material drying, be ground up, sieved and
The step of allotment, easy to operate, reproducible and recovery rate are high.The Cortex Phellodendri reference extract being prepared with this preparation method
Because being that multiple or different batches extracts deploy, overcome because Chinese medicine control medicinal material is by the place of production, growing environment
Influence, it is difficult to ensure that per batch of uniform quality defect, to ensure that the one of the Cortex Phellodendri reference extract of different batches
Cause property；Simultaneously by adding solvent appropriate, guarantee the consistency of extract and medicinal ingredient；Choose representative medicine
For material as raw material, obtained product is representative, and the quality of medicinal material to be detected more can be intuitively reflected as reference extract；
By adding auxiliary material, guarantee that extract character is stable, uniform and easy to use.Using Cortex Phellodendri reference extract of the invention
It is not less than 30% containing jamaicin in the finally obtained reference extract of preparation method.Further preferably, the content of jamaicin >=
34%.And reference extract TLC Fingerprints, HPLC finger-print are corresponding with medicinal material consistent；And with control medicinal material phase
Than easy to use, reference extract, which only needs simply to dissolve, be can be used without cumbersome extraction process.The present invention is also
Provide discrimination method or the Cortex Phellodendri or containing Cortex Phellodendri/Cortex Phellodendri effective component medicinal material or Chinese materia medica preparation of medicinal material or Chinese materia medica preparation
Method of quality control not only can carry out Qualitive test to medicinal material or Chinese materia medica preparation, but also it is even fixed to can be also used for sxemiquantitative
Amount analysis.
Detailed description of the invention
Fig. 1 phellodendron extract object reference substance TLC chromatogram S: jamaicin；1-3: extract reference substance)
Fig. 2 Cortex Phellodendri medicinal material and reference extract TLC chromatogram, 1,2: Cortex Phellodendri-ERS；3-14: Cortex Phellodendri medicinal material
Fig. 3 phellodendron extract reference substance HPLC-DAD chromatogram (S: jamaicin)
Fig. 4 Cortex Phellodendri medicinal material and reference extract HPLC-DAD chromatogram Cortex Phellodendri ERS and 6 batches of medicinal material HPLC map (blues
For ERS, other are medicinal material) Cortex Phellodendri-ERS is Cortex Phellodendri reference extract
Fig. 5 is the preparation method flow chart of Cortex Phellodendri reference extract.
Specific embodiment
With reference to the accompanying drawing, specific embodiments of the present invention will be described in detail, it is to be understood that guarantor of the invention
Shield range is not limited by the specific implementation.
Unless otherwise explicitly stated, otherwise in entire disclosure and claims, term " includes " or its change
Changing such as "comprising" or " including " etc. will be understood to comprise stated element or component, and not exclude other
Element or other component parts.
Embodiment 1, Cortex Phellodendri reference extract of the present invention replace the comparative test of control medicinal material
With thin-layered chromatography and high performance liquid chromatography respectively using chemical reference substance and Cortex Phellodendri reference extract as reference
Substance analyzes commercially available medicinal material.
1 thin-layer chromatography
1.1 sample preparation
Chemical reference substance solution: precision weighs jamaicin, and the solution of 0.1mg/ml is configured to methanol；
Cortex Phellodendri reference extract solution: precision weighs the precision that embodiment 3 is prepared and weighs phellodendron extract reference substance,
It is added methanol ultrasonic extraction 15 minutes, constant volume is configured to the solution of 2.5mg/ml concentration, by 0.45 μm of filter membrane, filtrate is taken to make
For extract reference substance solution.
Medicinal material test solution: Cortex Phellodendri medicinal powder (crossing No. two sieves) 0.2g is taken, methanol 20ml is added, ultrasonic extraction 30 is divided
Clock, filtration, takes filtrate as test solution.Pharmacy's medicinal material is bought from Guangzhou, and Cortex Phellodendri 1-14 is respectively as follows:
The detection of 1.2 thin-layer chromatographys
Testing conditions are as follows:
Lamellae: TLC G60 prefabricated board (Merck)
Point sample: 0.5 μ l, the long 6mm of ribbon point sample.
Solvent: butyl acetate: methanol: isopropanol: ammonium hydroxide=5:2:1.5:1.2
Expansion mode: the side double flute expansion cylinder (20cm*10cm) adds solvent 10ml, and it is small that two 10ml are placed in the other side
Beaker, each 10ml concentrated ammonia liquor test solution that is added pre-equilibrate 15 minutes jointly
* solvent usage amount: concentrated ammonia liquor usage amount=1:2
* the fixed sample-loading amount of the small beaker of each 10ml is 10ml concentrated ammonia liquor test solution, then the specification of the developing tank used is
When 10*10cm, it is only necessary to place the small beaker of a 10ml
The drying of load sample lamellae: the lamellae after point sample sets phosphorus pentoxide vacuum desiccator 2 hours, guarantees lamellae
Drying.It inspects: being placed under UV366nm and inspect.
Testing result: as shown in Figure 1, to inspect thin-layer chromatogram (T:17 DEG C, RH:37%) under UV366nm, Fig. 2 is
Thin-layer chromatogram (T:23 DEG C, RH:65%) is inspected under UV366nm
Interpretation of result: by Fig. 1,2 it is found that Berberine hydrochloride, Cortex Phellodendri reference extract and medicinal material (Cortex Phellodendri 1- Cortex Phellodendri 14) exist
Same position can show identical spot, find out that Cortex Phellodendri reference extract and medicinal material (Cortex Phellodendri 1-14) have height from map
Consistency.
2 high performance liquid chromatography
2.1 sample preparation
Chemical reference substance solution: precision weighs jamaicin 5mg, and the solution of 0.1mg/ml is configured to methanol；
Cortex Phellodendri reference extract solution: precision weighs the Cortex Phellodendri control that embodiment 3 is prepared and extracts, and methanol-salt is added
Sour (volume ratio of 100:1), ultrasonic (500w) are handled 30 minutes, are let cool, and are configured to the solution that concentration is 0.24mg/ml, mistake
0.22 μm of filter membrane, take subsequent filtrate to get.
Medicinal material test solution: Cortex Phellodendri medicinal powder (crossing No. two sieves) 0.2g is taken, methanol 20ml is added, ultrasonic extraction 30 is divided
Clock, filtration, takes filtrate as test solution.Pharmacy's medicinal material is bought from Guangzhou, and Cortex Phellodendri 1-6. is respectively as follows:
2.2 high performance liquid chromatography detection
High performance liquid chromatography testing conditions are as follows:
Chromatographic apparatus: Agilent HPLC1260 high performance liquid chromatograph
Detector: Agilent DAD detector
Chromatographic column: Kromasil 100-5C184.6*250mm, 5 μm.
Mobile phase: (A) acetonitrile；(B) -0.3% triethylamine of 0.3% phosphoric acid (1:1)
Eluent gradient:
The preparation of Cortex Phellodendri reference extract solution takes Cortex Phellodendri reference extract appropriate, accurately weighed, and methanol-hydrochloric acid is added
The mixed solution ultrasonic treatment (power 250W, frequency 40kHz) of (100:1) 30 minutes, lets cool, and being configured to concentration is 0.24mg/
The solution of ml, cross 0.45um filter membrane, take subsequent filtrate to get.
The preparation precision of reference solution weighs jamaicin reference substance, and the solution of 0.1mg/ml is configured to methanol.
Medicinal material test solution takes Cortex Phellodendri medicinal powder (crossing No. two sieves) 0.2g, methanol 20ml is added, ultrasonic extraction 30 is divided
Clock, filtration, takes filtrate as test solution.Pharmacy's medicinal material is bought from Guangzhou, and Cortex Phellodendri 1-6 is respectively as follows:.
Accurate absorption reference solution, control mention extract solution and each 10 μ l of test solution to measuring method respectively, inject
Liquid chromatograph, measurement, record chromatogram to get.
The identical chromatographic peak of corresponding object of reference chromatographic peak retention time should be presented in test article fingerprint respectively.
High performance liquid chromatography testing result:
Measurement, records chromatogram to get Fig. 3, S- jamaicin, HPLC finger-print stacking chart shown in Fig. 4, from top to bottom
1-7 respectively corresponds Cortex Phellodendri 1, Cortex Phellodendri 2, Cortex Phellodendri 3, Cortex Phellodendri 4, Cortex Phellodendri 5, Cortex Phellodendri 6, and ERS corresponds to Cortex Phellodendri reference extract.
16 batches of medicinal material assays of table:
| % | Jamaicin | RSD (n=2) |
| Cortex Phellodendri medicinal material 1 | 9.03 | 0.86 |
| Cortex Phellodendri medicinal material 2 | 9.57 | 0.17 |
| Cortex Phellodendri medicinal material 3 | 9.62 | 0.07 |
| Cortex Phellodendri medicinal material 4 | 10.73 | 0.31 |
| Remove the peel CORTEX PHELLODENDRI CHINENSE | 8.96 | 1.82 |
| Belt leather CORTEX PHELLODENDRI CHINENSE (after peeling) | 6.97 | 0.13 |
As seen from Figure 4, the finger-print of 6 medicinal material of Cortex Phellodendri 1- Cortex Phellodendri has high consistency, thus establishes Cortex Phellodendri
The finger-print common pattern of medicinal material, utilizes Chinese Pharmacopoeia Commission's similarity evaluation
(2012.130723 version) calculates, and carries out similarity analysis to all samples.
According to the result of TLC and HPLC finger-print it is found that being examined to the Cortex Phellodendri reference extract using thin-layered chromatography
The corresponding raw medicinal material of the chromatogram measured is consistent, more precisely, uses thin layer to the Cortex Phellodendri reference extract
The fluorescence spot and use high performance liquid chromatography detection at Berberine hydrochloride position in the chromatogram that chromatography detects
Chromatographic peak of the obtained chromatogram at Berberine hydrochloride position distinguishes corresponding chemical reference substance or its corresponding raw material
Medicinal material is consistent, therefore Cortex Phellodendri reference extract of the invention can be applied to the Qualitive test of medicinal material or Chinese materia medica preparation, such as to medicine
The Berberine hydrochloride ingredient of material or Chinese materia medica preparation carries out qualitative analysis.
According to HPLC measurement result and statistical analysis, the Cortex Phellodendri medicinal materials fingerprint common pattern and Cortex Phellodendri medicinal material of foundation
Similarity it is high, and the similarity pole of Cortex Phellodendri reference extract and Cortex Phellodendri medicinal materials fingerprint common pattern of the invention
Height, this explanation carry out the assay of Berberine hydrochloride using Cortex Phellodendri reference extract of the invention to medicinal material or Chinese materia medica preparation
Reliability is high, therefore Cortex Phellodendri reference extract of the invention can be applied to the sxemiquantitative discriminatory analysis of medicinal material or Chinese materia medica preparation,
Can also be applied to Cortex Phellodendri and containing in the control of the quality of Cortex Phellodendri/Cortex Phellodendri effective component medicinal material or Chinese materia medica preparation, for example, to medicinal material or
The Berberine hydrochloride ingredient of Chinese materia medica preparation carries out sxemiquantitative discriminatory analysis, or to Cortex Phellodendri and containing Cortex Phellodendri/Cortex Phellodendri effective component
Medicinal material or Chinese materia medica preparation carry out half-quantitative detection and then control its quality.
The character analysis of 2 Cortex Phellodendri reference extract of embodiment
1. apparent state: the Cortex Phellodendri reference extract that embodiment 3 obtains is yellow powder.
2. determination of moisture: being carried out according to 2010 editions Chinese Pharmacopoeia annex IX G (hypobaric drying method).Testing result is Cortex Phellodendri
Reference extract water content is 5.41%.
3. uniformity test: preparing 6 batch Cortex Phellodendri reference extracts according to the method for embodiment 1, each batch is small after measured
Bark of a cork tree alkali, thin-layer chromatography testing result difference very little；Through high performance liquid chromatography detection, its content of berberine is deploying standard model
Within enclosing.Therefore the Cortex Phellodendri reference extract being prepared using the preparation method of Cortex Phellodendri reference extract of the invention is consistent
Property is very good.
4. stability test:
The test sample for taking the Cortex Phellodendri reference extract prepared according to the method for embodiment 1 of 10 different batches, according to state
The relevant regulations of " requirement of national drug standards substance Development Techniques " that pharmacopoeia commission, family works out, inspection target includes character
And dissolubility.
Test sample opening is set in suitable clean container, is placed 10 days at a temperature of 60 DEG C, is sampled in the 5th day and the 10th day,
It is detected by stability high spot reviews project.
The results show that before and after hot test test sample character without significant change, before and after hot test thin-layer chromatogram also without
Significant change, dissolution rate measurement result carry out Independent samples t-test with SPSS, and calculated result P > 0.05 illustrates no conspicuousness
Difference.
The preparation of 3 Cortex Phellodendri reference extract of embodiment
Instrument and reagent: the semi-automatic point sample instrument of 5 thin-layer chromatography of Linomat, the full-automatic point sample instrument of 4 thin-layer chromatography of ATS, thin
Layer chromatography double flute expansion cylinder, the colour developing of Chromatogram Immersion Device III chromatography dipping tank and TLC
Visualizer thin-layer chromatography video camera (Switzerland, CAMAG).
Agilent 1260series high performance liquid chromatograph is furnished with DAD detector (U.S., Agilent
Technologies)。
Methanol, ethyl alcohol, isopropanol are to analyze pure (Tianjin great Mao chemical reagent factory).Toluene, ethyl acetate, acetic acid fourth
Ester, concentrated ammonia liquor and triethylamine are to analyze pure (Guangzhou Chemical Reagent Factory).
Berberine hydrochloride (also referred to as jamaicin) indicates purity >=98%, (Sichuan Wei Keqi Biotechnology Co., Ltd)；
It is envisaged for preparing the medicinal material of reference extract: Cortex Phellodendri.
Cortex Phellodendri medicinal material 6 batches buyings are in the major pharmacy in Guangzhou and medicinal material market
The preparation method of Cortex Phellodendri reference extract of the present invention is present embodiments provided, method flow diagram is as shown in Figure 5.
One, it extracts
It chooses Cortex Phellodendri medicinal material and powder is made, medicinal powder crosses No. two sieves (850 ± 29 μm, 24 mesh), its quality is then added
The methanol (i.e. solid-to-liquid ratio is 1:10 (w/V)) of 10 times of volumes, middling speed is qualitative after ultrasonic (power 500w, room temperature) extracts 30min
Filter paper filtering, collects filter residue 1 and filtrate 1, and filter residue 1 is extracted secondary again by same procedure, and will extract secondary collection again
Filtrate merges with filtrate 1 Ji Wei extracting solution, and extracting solution solvent evaporated is got dry extract.
Two, phellodendron extract is prepared
After dry cream is dissolved completely with the methanol of dry cream 2-5 times (w/v), then plus 40% micro mist silicon of dry cream weight
20% medical starch of glue and dry cream weight, is evaporated with Rotary Evaporators, crushed 200 meshes, obtain phellodendron extract.
Three, extract analyte detection: 6 batches of phellodendron extracts of preparation are detected according to the detection method of test example one, and testing result is such as
Shown in table 2.
The component content table of 2 phellodendron extract of table
| % | Jamaicin | RSD (n=4) |
| Cortex Phellodendri reference extract 1 | 37.30 | 3.15 |
| Cortex Phellodendri reference extract 2 | 39.03 | 1.04 |
| Cortex Phellodendri reference extract 3 | 38.40 | 1.15 |
| Cortex Phellodendri reference extract 4 | 42.48 | 2.06 |
| Remove the peel CORTEX PHELLODENDRI CHINENSE | 37.31 | 2.50 |
| Belt leather CORTEX PHELLODENDRI CHINENSE (after peeling) | 34.46 | 1.40 |
Four, it deploys
The phellodendron extract of 6 batches of step 3 is mixed in a certain ratio and blends Cortex Phellodendri reference extract, is deployed
Standard are as follows: it is about 1:4 (g/g) that final products, which correspond to crude drug ratio, should make final Cortex Phellodendri reference extract Berberine
Content >=30%,
Component content in 3 Cortex Phellodendri reference extract of table
Claims (10)
1. a kind of Cortex Phellodendri reference extract, which is characterized in that the extract is yellow dried powder, is by containing weight percent
It is formed than phellodendron extract 67-84% and auxiliary material 16-33%, wherein phellodendron extract Berberine content is not less than 30%.
2. Cortex Phellodendri reference extract as described in claim 1, which is characterized in that wherein phellodendron extract Berberine content is not
Lower than 34%.
3. Cortex Phellodendri reference extract as described in claim 1, which is characterized in that the auxiliary material is superfine silica gel powder, medical starch
Middle one or two.
4. a kind of Cortex Phellodendri reference extract as described in claim 1, which is characterized in that be to be prepared by the following method
:
(1) Cortex Phellodendri dry cream: the Cortex Phellodendri medicinal powder methanol ultrasonic extraction of separate sources and batch is taken, filtrate, filtrate are filtered to obtain
It is evaporated to obtain Cortex Phellodendri dry cream；
(2) phellodendron extract: Cortex Phellodendri dry cream is dissolved in methanol, adds auxiliary material and is evaporated and is ground up, sieved up to phellodendron extract；
(3) Cortex Phellodendri reference extract: carrying out allotment for separate sources or the phellodendron extract of batch and blend, and obtains Cortex Phellodendri control and mentions
Take object.
5. a kind of Cortex Phellodendri reference extract as claimed in claim 4, which is characterized in that increase different Cortex Phellodendri medicines before step (1)
The screening step in material source: by separate sources or the raw medicinal material of batch with thin-layered chromatography and/or high performance liquid chromatography into
Row detection；Then the raw medicinal material of separate sources or batch is obtained by thin-layered chromatography and/or high performance liquid chromatography
Chromatogram and/or the content of berberine of measurement be compared analysis, reject the raw material of the content exception of chromatogram and/or measurement
Medicinal material retains the preparation of the Cortex Phellodendri dry cream of the consistent raw medicinal material progress next step of content of chromatogram and/or measurement.
6. a kind of Cortex Phellodendri reference extract as claimed in claim 4, which is characterized in that further include between step 2 and step 3 with
The step of thin-layered chromatography and/or high performance liquid chromatography detect the jamaicin of the phellodendron extract of different batches.
7. a kind of Cortex Phellodendri reference extract as claimed in claim 4, which is characterized in that Cortex Phellodendri medicinal powder and first in step 1
The w/v of alcohol is 1:8~1:50；The ultrasonic time is 30~60min, and power is 100W~3kW；Methanol in step 2
Dosage is 2-5 times of dry cream weight；Allotment described in step 3 refers specifically to: separate sources or the phellodendron extract of batch are carried out
Allotment, should make to correspond to crude drug ratio 1:(2-5 in final Cortex Phellodendri reference extract) g/g, wherein content is small in terms of wt%
Content >=34% of bark of a cork tree alkali.
8. a kind of Cortex Phellodendri reference extract as claimed in claim 6, which is characterized in that the thin-layer chromatography of the phellodendron extract
Method and/or high effective liquid chromatography for detecting, include the following steps:
Wherein thin-layered chromatography includes the following steps:
(1) chemical reference substance solution: precision weighs jamaicin, and the solution of 0.1mg/ml is configured to methanol；
(2) phellodendron extract solution: accurate phellodendron extract is ultrasonically treated with methanol, constant volume, and being made into concentration is that 2.5mg/ml is molten
Liquid crosses 0.22 μm of filter membrane to obtain the final product；
(3) the thin-layered chromatography testing conditions are as follows:
Lamellae: G60 prefabricated board；
Point sample: 0.5 μ l, the long 6mm of ribbon point sample；
Solvent: butyl acetate: methanol: isopropanol: ammonium hydroxide=5:2:1.5:1.2；
Expansion mode: double flute expansion cylinder, side add solvent, and the other side is placed concentrated ammonia liquor test solution and pre-equilibrated jointly 15 minutes, open up
Open agent usage amount: concentrated ammonia liquor usage amount=1:1-2；
The drying of plate: the lamellae after point sample is placed in phosphorus pentoxide vacuum desiccator, guarantees the drying of lamellae；
It inspects: being placed under UV366nm and inspect；
Wherein high performance liquid chromatography includes the following steps:
(1) preparation of chemical reference substance solution: precision weighs jamaicin, and the solution of 0.1mg/ml is configured to methanol；
(2) preparation of phellodendron extract test solution: taking phellodendron extract that methanol-hydrochloric acid (100:1) is added, ultrasonic treatment,
It lets cool, is configured to the solution that concentration is 0.24mg/ml, filtration takes subsequent filtrate to get phellodendron extract solution；
(3) chromatographic condition:
The high performance liquid chromatography testing conditions:
Chromatographic apparatus: high performance liquid chromatograph is furnished with DAD detector
Chromatographic column: C18Column (250mm × 4.6mm, 5 μm)；
Mobile phase: A- acetonitrile, B- -0.3% triethylamine 1:1 containing 0.3% phosphoric acid；
Gradient elution program:
Detection wavelength 270nm, DAD detector；Flow velocity 0.8ml/min；10 μ l of sample volume；25 DEG C of column temperature；
(4) detection method: precision draws each 10 μ l of test solution, injects liquid chromatograph, records chromatogram and calculates barberry
Alkali content.
9. a kind of Cortex Phellodendri reference extract as described in claim 1, which is characterized in that the Cortex Phellodendri reference extract is using thin
The fluorescence spot for the jamaicin in chromatogram that layer chromatography detects and the color detected using high performance liquid chromatography
It is consistent that chromatographic peak of the spectrogram at jamaicin position should distinguish corresponding chemical reference substance or its corresponding raw medicinal material.
10. the discrimination method or Cortex Phellodendri of a kind of Cortex Phellodendri medicinal material or Chinese materia medica preparation or the medicinal material or Chinese medicine system of the effective component containing Cortex Phellodendri
The method of quality control of agent, which is characterized in that by Cortex Phellodendri reference extract described in claim 1, sample to be tested with thin layer
Chromatography and/or high performance liquid chromatography are detected, and comparison differentiates,
Wherein thin-layered chromatography includes the following steps:
(1) chemical reference substance solution: precision weighs jamaicin, and the solution of 0.1mg/ml is configured to methanol；
(2) Cortex Phellodendri reference extract solution: accurate Cortex Phellodendri reference extract is ultrasonically treated with methanol, constant volume, is made into concentration and is
2.5mg/ml solution crosses 0.22 μm of filter membrane to obtain the final product；
(3) sample to be tested: medicinal material test solution or preparation containing Cortex Phellodendri: taking sample, and methanol ultrasound is added, takes out after ultrasound, from
The heart takes supernatant to cross 0.22um filter membrane up to medicinal material test solution；
(4) the thin-layered chromatography testing conditions are as follows:
Lamellae: G60 prefabricated board；
Point sample: 0.5 μ l, the long 10mm of ribbon point sample；
Solvent: butyl acetate: methanol: isopropanol: ammonium hydroxide=5:2:1.5:1.2；
Expansion mode: double flute expansion cylinder, concentrated ammonia liquor test solution pre-equilibrate, solvent usage amount: concentrated ammonia liquor usage amount=1:(1-2)；
The drying of lamellae: the lamellae after point sample is placed in phosphorus pentoxide vacuum desiccator, guarantees the drying of lamellae；
It inspects: being placed under UV366nm and inspect；
Wherein high performance liquid chromatography includes the following steps:
(1) preparation of chemical reference substance solution: precision weighs jamaicin, and the solution of 0.1mg/ml is configured to methanol；
(2) preparation of Cortex Phellodendri reference extract solution: taking Cortex Phellodendri reference extract that methanol-hydrochloric acid (100:1) is added, at ultrasound
Reason, lets cool, and is configured to the solution that concentration is 0.24mg/ml, and filtration takes subsequent filtrate to get Cortex Phellodendri reference extract solution；
(3) sample to be tested test solution: Cortex Phellodendri medicinal powder or preparation containing Cortex Phellodendri, it is accurately weighed, it sets in stuffed conical flask, essence
Close addition methanol, close plug, ultrasound are taken out, are let cool, and methanol is added to shake up to scale, filtration to get；
(4) chromatographic condition:
The high performance liquid chromatography testing conditions:
Chromatographic apparatus: high performance liquid chromatograph is furnished with DAD detector
Chromatographic column: C18Column (250mm × 4.6mm, 5 μm)；
Mobile phase: A- acetonitrile, the aqueous solution (1:1) of B- -0.3% triethylamine Han 0.3% phosphoric acid；
Gradient elution program:
Detection wavelength 270nm, DAD detector；Flow velocity 0.8ml/min；10 μ l of sample volume；25 DEG C of column temperature；
(5) detection method: precision draws each 10 μ l of medicinal material test solution, injects liquid chromatograph, records chromatogram and calculating
Content of berberine.
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