CN103604879B - Detection method of polystictus versicolor and preparation containing polystictus versicolor - Google Patents
Detection method of polystictus versicolor and preparation containing polystictus versicolor Download PDFInfo
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Abstract
The invention discloses a detection method of polystictus versicolor and a preparation containing polystictus versicolor. According to the method, monosaccharide fragments obtained after polysaccharides of polystictus versicolor and the preparation containing polystictus versicolor are hydrolyzed are detected by adopting ultra performance liquid chromatography or high performance liquid chromatography while mannitose, glucose, galactose, fucose and other monosaccharide fragments are taken as evaluating indicators, and a quality control and evaluation method for measuring characteristic spectra and content of the monosaccharide fragments is established. The method can be used for quality control and evaluation of a polystictus versicolor crude drug and a preparation containing the polystictus versicolor crude drug, a polystictus versicolor extract or a polystictus versicolor effective part. The method is used for evaluating the quality of the polystictus versicolor crude drug and the preparation by controlling the monosaccharide fragments obtained after polysaccharide of the polystictus versicolor and the preparation containing polystictus versicolor are hydrolyzed. The method is strong in specificity, high in accuracy, easy and simple to operate, easy to control and higher in practical value, and can be used for effectively controlling the product quality.
Description
Technical field
The present invention relates to a kind of Coriolous Dersicolor (Fr.) Quel and the detection method of the preparation containing Coriolous Dersicolor (Fr.) Quel, be specifically related to Coriolous Dersicolor (Fr.) Quel medical material and contain
The quality control of the preparation of Coriolous Dersicolor (Fr.) Quel or Coriolous Dersicolor (Fr.) Quel effective site or Coriolous Dersicolor (Fr.) Quel effective part group and evaluation methodology.
Background technology
Coriolous Dersicolor (Fr.) Quel is that On Polyporaceae Corilus versicolor Quel. Coriolus versicolor (L.ex Fr.) Quel is dried son
Entity.The Eastern Han Dynasty Shennong's Herbal have been described with Coriolous Dersicolor (Fr.) Quel treat cancer, Compendium of Material Medica record Coriolous Dersicolor (Fr.) Quel have calm the nerves, benefit
Longevity, profit joint, hard muscles and bones, control deaf effect.Modern study shows, the krestin extracted from Coriolous Dersicolor (Fr.) Quel is for tumor patient
Auxiliary treatment, can significantly improve the life quality of cancer patient, strengthens immunity, reduces secondary infection, alleviates slight illness, improves cancer
The loss of appetite effect of patient.Owing to the unique activity of krestin has the latentest with low toxic effect in clinical practice
Power, for krestin constituents exploitation health product, medicine day by day increase, 1977, the Coriolous Dersicolor (Fr.) Quel of extraction purification from Coriolous Dersicolor (Fr.) Quel
Polysaccharide (PSK), is developed to PTS Krestin in Japan；1998, many with the Coriolous Dersicolor (Fr.) Quel that extraction purification from Coriolous Dersicolor (Fr.) Quel obtains
Glycopeptide (PSP) is the formal production approval number that the Polysaccharide-peptide capsule of main component formally obtains China's Ministry of Public Health, the most
Commercialized product has polysaccharide-peptide, Coriolous Dersicolor (Fr.) Quel fungus capsule, manyzoned polypore gantai sheet, manyzoned polypore gantai capsule etc..At present, domestic total polysaccharides is containing measuring
Determining mainly to take ultraviolet spectrophotometry to carry out, classical color development system has phend-sulphuric acid (λ max=490nm) and anthrone-sulfur
Acid system (λ max=630nm), all selecting anhydrous glucose is reference substance.Also have and use titration measuring total polysaccharides content, often
With having Fei Linshi titrimetry and Indirect iodimetry.Total polysaccharides content in 2010 editions " Chinese Pharmacopoeias ", in Coriolous Dersicolor (Fr.) Quel medical material
Measure and just use indirect iodometric processes.Owing to Coriolous Dersicolor (Fr.) Quel medical material and preparation thereof are mostly colored samples, this type of method is applied to measure
During Coriolous Dersicolor (Fr.) Quel total polysaccharides in sample, the interference factor of method is many, and result precision, specificity are poor.Quality to polysaccharide abroad
Controlling many employing high productivity computing methods and measure polysaccharide molecular weight and distribution thereof, this is also to one of polyose Quality Control means,
Its sugar product quality control more much higher to purity is preferable, the true and false of energy this product of effectively evaluating.Domestic Coriolous Dersicolor (Fr.) Quel series products is also
There is employing high productivity computing method to measure polysaccharide molecular weight and distribution thereof, evaluate the quality of this series products, the method energy with this
Control the quality of product to a certain extent, but for the main Coriolous Dersicolor (Fr.) Quel medical material containing polysaccharide composition and the quality control of preparation thereof
Method is less, owing to they contained effective polysaccharide are big classes, is difficult to evaluate its polyoses content, range of molecular weight distributions
Medical material and the true and false of the quality of the pharmaceutical preparations thereof and quality, this evaluation method can not reflect the inherent quality change of they entirety, have one
Fixed irrationality and the most scientific, and specificity is the most poor.Meanwhile, polysaccharide molecule composition in Coriolous Dersicolor (Fr.) Quel medical material and preparation thereof, empty
Between structure different, and the molecular weight of the sample polysaccharide recorded with corresponding reference substance, the not absolute value of molecular weight.
Summary of the invention
It is an object of the invention to provide the Coriolous Dersicolor (Fr.) Quel that a kind of accuracy is high, specificity is strong, easy and simple to handle and the system containing Coriolous Dersicolor (Fr.) Quel
The detection method of agent, in order to more preferably carry out quality control and evaluation.
The present invention, according to the main contents that the monosaccharide composition of Coriolous Dersicolor (Fr.) Quel medical material and preparation polysaccharide thereof is its primary structure, is that it is empty
Between the basis of structure, it is necessary first to its polysaccharide is carried out after complete acid hydrolysis is monosaccharide material, the one-tenth of monosaccharide contained therein
Divide, the mutual ratio of each monosaccharide components content should have species uniqueness and individual comparability, therefore, carries out for it
Chromatography, the chromatograph collection of illustrative plates obtained has stronger specificity.Use liquid chromatograph separation detection technique, analyze Coriolous Dersicolor (Fr.) Quel medicine
In material and preparation thereof, the monosaccharide composition of polysaccharide, sets up corresponding characteristic spectrum, by using monosaccharide component characteristic spectrum in polysaccharide
Relative retention time and relative peak area evaluate this medical material true and false, this quality control and evaluation methodology have stronger exclusive
Property and novelty.Meanwhile, on the basis of characteristic spectrum, set up monosaccharide component quantitative determination, and formulate each content limit, pass through
Control to monosaccharide quality can control the content of polysaccharide indirectly, evaluates the quality of quality of medicinal material, this matter with this method of quality control
Amount controls and evaluation methodology has higher accuracy and novelty.
The technical scheme is that
Taking Coriolous Dersicolor (Fr.) Quel medical material 3～6g(and add water in right amount, heating and refluxing extraction, extracting solution is centrifuged, measures appropriate extracting solution, decompression
Be concentrated into appropriate) or preparation 0.5～1g(add water and make dissolving), add the dehydrated alcohol of 3～6 times of volumes, point take precipitation, precipitation adds
Water dissolution, and add dilute sulfuric acid (concentration is 10%), 100-120 DEG C is heated to reflux hydrolysis 5-6 hour, and after hydrolysis, solution adjusts pH extremely
Neutral also constant volume, Aspirate supernatant, put in tool plug test tube, add NaOH solution and the methanol solution of PMP, derive in 60～80 DEG C
Change reaction 30-60min, after reaction completely, tune PH is to neutral and constant volume, takes supernatant and adds the shaking extraction of equal-volume chloroform, abandons
Going organic solution, repetitive operation 3 times, aqueous solution is evaporated to use acetonitrile constant volume in right amount, as need testing solution；Take D-manna
Sugar, D-Glucose, galactose and L-fucose reference substance are appropriate, are dissolved in water, by the deriving method system of above-mentioned need testing solution
Standby mixing reference substance solution, precision draws mixing reference substance solution and need testing solution respectively, inject high performance liquid chromatograph or
Ultra Performance Liquid Chromatography instrument, measures each contents of monosaccharides.Wherein Ultra Performance Liquid Chromatography testing conditions is: with octadecylsilane key
Conjunction silica gel is filler, and flow velocity is 0.3～1.0 ml/min；Detection wavelength 245nm；With acetonitrile as mobile phase A, delay with phosphoric acid
Rush saline solution and (weigh K2HPO44.4g, the 800mL that adds water dissolve, and add phosphoric acid and adjust pH to 6.5, and are diluted with water to 1000mL) for flowing
Dynamic phase B, eluent gradient eluting table:
High performance liquid chromatography testing conditions is: with octadecylsilane chemically bonded silica as filler, and flow velocity is 0.8～1.5
Ml/min；Detection wavelength 245nm；With acetonitrile as mobile phase A, with phosphate buffered saline(PBS) as Mobile phase B, eluent gradient
Eluting table:
In the chromatogram of gained, the characteristic spectrum of need testing solution should present mannose, glucose, galactose, rock algae
4 characteristic peaks of sugar, it should be consistent with the retention time at corresponding object of reference peak respectively, with wherein any one object of reference peak phase
Corresponding peak is S peak, calculates each characteristic peak and the relative retention time at S peak, relative peak area, and its relative retention time should be on rule
Definite value ± 5～10% within the scope of, relative peak area can fluctuate 10～30%.
Coriolus Versicolor P.E. real and fake discrimination:
The peak corresponding with galactose object of reference peak, for S peak, calculates the relative retention time of each characteristic peak and S peak, its phase
To retention time should setting ± 5% within the scope of, it is stipulated that be worth and be: 0.5(mannose), 0.9(glucose), 1.0(gala
Sugar), 1.1(fucose)；
The peak corresponding with galactose object of reference peak, for S peak, calculates the relative peak area of each characteristic peak and S peak, and it is relative
Peak area should setting ± 30% within the scope of, relative peak area setting is: 0.6(mannose), 16.5(glucose),
1.0(galactose), 0.4(fucose).
Coriolous Dersicolor (Fr.) Quel medical material real and fake discrimination:
The peak corresponding with galactose object of reference peak, for S peak, calculates the relative retention time of each characteristic peak and S peak, its phase
To retention time should setting ± 5% within the scope of, it is stipulated that be worth and be: 0.5(mannose), 0.9(glucose), 1.0(gala
Sugar), 1.1(fucose)；
The peak corresponding with galactose object of reference peak, for S peak, calculates the relative peak area of each characteristic peak and S peak, and it is relative
Peak area should setting ± 30% within the scope of.Relative peak area setting is: 0.8(mannose), 22.5(glucose),
1.0(galactose), 0.3(fucose)；
Each characteristic peak exists, and relative retention time and relative peak area meet the requirements, and can evaluate Coriolous Dersicolor (Fr.) Quel and containing cloud with this
The true and false of the preparation of sesame.
Quality evaluation:
A, Coriolous Dersicolor (Fr.) Quel medical material in terms of mannose, glucose, galactose, fucose total amount, must not be less than 4.0% containing krestin；
B, Coriolus Versicolor P.E., must not be less than containing krestin in terms of mannose, glucose, galactose, fucose total amount
25%。
The good effect that the present invention produces is: the present invention uses and " extracts crude polysaccharides-polysaccharide hydrolysis-derivatization-characteristic pattern
Spectrum-quantitatively " quality control model, evaluate Coriolous Dersicolor (Fr.) Quel medical material and the quality of preparation thereof, this quality control and evaluation methodology and existing right
Medical material containing polysaccharide is compared with the method for quality control of preparation, has more stronger specificity and novelty.At characteristic spectrum
On the basis of, set up monosaccharide component quantitative determination, by the control of monosaccharide quality being controlled indirectly the content of polysaccharide, with this quality
Control method evaluates Coriolous Dersicolor (Fr.) Quel and the quality of the quality of the pharmaceutical preparations containing Coriolous Dersicolor (Fr.) Quel, and this quality control and evaluation methodology have higher accuracy
And novelty.Thinking of the present invention and pattern can be for providing, containing polysaccharide Chinese medicine and preparation, the quality control that specificity is strong, accuracy is high
System and evaluation methodology.
Accompanying drawing explanation
Fig. 1 is the UPLC-PDA chromatogram of standard monosaccharide mixing reference substance (10 kinds of monosaccharide)；
Fig. 2 is the HPLC-DAD chromatogram of standard monosaccharide mixing reference substance (5 kinds of monosaccharide)；
Fig. 3 is the UPLC-PDA chromatogram of blank solvent；
Fig. 4 is the HPLC-DAD chromatogram of the monosaccharide composition of polysaccharide in Coriolous Dersicolor (Fr.) Quel medical material；
Fig. 5 is the UPLC-PDA chromatogram of the monosaccharide composition of polysaccharide in Coriolous Dersicolor (Fr.) Quel medical material；
Fig. 6 is the HPLC-DAD chromatogram of the monosaccharide composition of polysaccharide in Coriolus Versicolor P.E.；
Fig. 7 is the UPLC-PDA chromatogram of the monosaccharide composition of polysaccharide in Coriolus Versicolor P.E..
Detailed description of the invention
The preparation method of the present invention and chromatographic condition and quality control and evaluation methodology are through substantial amounts of experiment sieving
The preferred plan obtained, following experiment example is used for further illustrating technical scheme and technique effect.
Experimental example 1: the characteristic spectrum research of Coriolous Dersicolor (Fr.) Quel medical material and preparation thereof
Due to the heteropolysaccharide that krestin is a big class, structure is complicated, at present still without the quality control side that specificity is strong
Method, it is impossible to differentiate different types of polysaccharide.And the method for the present invention is stable, favorable reproducibility, specificity is strong, can distinguish dissimilar
Polysaccharide, can be used for controlling to evaluate Coriolous Dersicolor (Fr.) Quel medical material and the quality of preparation thereof.Result of the test is as follows:
1, with reference to the preparation of solution
Preparation with reference to solution accurately weighs standard substance monosaccharide in right amount, is configured to suitable monosaccharide standard substance with water dissolution
Liquid liquid.
Derivative with reference to solution measures above-mentioned reference solution in right amount, adds 0.5mLNaOH(0.5M), 0.8mLPMP methanol solution
(0.5mol/L), mixing, in 70 DEG C of derivative reaction 45min, adding hydrochloric acid (0.5mol/L) 0.7mL(pH value after letting cool is 4), turn
Moving to 10mL measuring bottle, and be diluted with water to scale, 5000rpm is centrifuged 10min, takes supernatant and adds the extraction of equal-volume chloroform, vibrate,
Centrifugal, discard lower floor's chloroform, be repeated 3 times, take upper water solution, obtain with reference to solution.
2, prepared by need testing solution
Weighing Coriolus Versicolor P.E. preparation 1.0g, the 5mL that adds water makes dissolving, adds 4 times amount ethanol, shakes up, centrifugal 10min,
Precipitation separation, and precipitate 3 times with absolute ethanol washing, precipitation adds 8mL water makes dissolving, then adds dilute sulfuric acid (weight concentration is 10%)
7mL, shakes up, and 100-120 DEG C is heated to reflux hydrolysis 5-6 hour, lets cool, and adjusts pH the most neutral by NaOH solution, and is transferred to 25mL
Measuring bottle, is diluted with water to scale, and centrifugal, precision measures supernatant 0.5mL, is derived by " derivative with reference to solution " method, to be obtained final product
Need testing solution.
The dehydrated alcohol consumption (1 times, 2 times, 3 times, 4 times, 5 times) of Chinese medicine Polyose extraction is investigated by the present invention, knot
Fruit selects the ethanol of 4 times amount effectively to be removed by the monosaccharide dissociated in sample.
The acid of Chinese medicine polysaccharide hydrolysis is investigated by the present invention, and result, without significant difference, selects sulphuric acid hydrolysis liquid more steady
Fixed, hydrolyze more complete.Meanwhile, to the consumption (5ml, 7ml, 9ml) of the dilute sulfuric acid that weight concentration is 10% and hydrolysis time, (4 is little
Time, 7ml, 9ml) investigated, to add 7 ml dilute sulfuric acid best results.
The present invention to the consumption (0.6 mL, 0.8 mL, 1.0mL) of derivative reagent PMP methanol solution and concentration (0.3 mol/L,
0.5 mol/L, 0.8mol/L) to investigate, result selects the PMP methanol solution best results of 0.8 mL and 0.5 mol/L.Simultaneously
Temperature (60 DEG C, 70 DEG C, 80 DEG C) and time (30min, 45min, 60min) to derivatization are all investigated, and result is selected
70 DEG C of derivative reaction 45min best results.
3, blank solution preparation takes blank reagent and prepares blank solution by " prepared by need testing solution " method.
4, chromatographic condition and system suitability
Instrument: waters UPLC, DAD detector；Chromatographic column: octadecylsilane chemically bonded silica chromatographic column (Kinetex
C18100A, 4.6 × 150mm, 2.6 μm), (weigh K with acetonitrile and phosphate2HPO44.4g, the 800mL that adds water dissolve, and add phosphoric acid
Adjust pH to 6.5, and be diluted with water to 1000mL) carry out gradient elution for flowing phase according to the form below；Column temperature: 35 DEG C；Detection wavelength:
245nm；Sample size: 2.0 μ l；Flow velocity: 0.9ml/min.
Precision is drawn with reference to solution and sample solution, each 2 L of blank solution respectively, injects Ultra Performance Liquid Chromatography instrument,
Under the conditions of this, each monosaccharide chromatographic peak reaches baseline separation, and blank reagent is noiseless.
The selection of the phase that 5, flows
Being investigated the gradient of flowing phase, result is optimal with selected gradient, 10 monosaccharide can be reached baseline and divide
From；And the flowing phosphate of phase and buffer solution are selected, wherein buffer solution pH is relatively big on chromatographic peak impact, result with
Selected condition pH value is 6.5 optimal.
6, Method validation
Instrument precision is tested: result need testing solution is with galactose for reference to solution, and it is relative that it respectively mainly comprises monosaccharide
Retention time RSD% is respectively as follows:
It respectively mainly comprises monosaccharide relative peak area RSD% and is respectively as follows:
Result shows that this instrument precision is preferable.
Test liquid stability test: result need testing solution, in 24 hours, respectively mainly comprises the relative retention time of monosaccharide
RSD is respectively as follows:
Result shows that the relative retention time stability mainly comprising monosaccharide is preferable.
Relative peak area RSD respectively mainly comprising monosaccharide is respectively as follows:
Result shows that the relative peak area stability mainly comprising monosaccharide is preferable.
Replica test: repeat test in this way, respectively mainly comprise relative retention time and the relative peak area of monosaccharide
RSD is respectively as follows:
Result shows to mainly comprise the relative retention time repeatability of monosaccharide preferably.
Result shows to mainly comprise the relative peak area repeatability of monosaccharide preferably.
7, characteristic spectrum determines
Through the test sample characteristic spectrum mensuration of too much batch Coriolus Versicolor P.E. preparation, wherein test sample collection of illustrative plates should present 4
Characteristic spectrum, the peak corresponding with galactose object of reference peak, for S peak, calculates the relative retention time of each characteristic peak and S peak, Ying
Within the scope of the 5% of setting, relative retention time setting is: 0.5(peak 1), 0.9(peak 2), 1.00(peak 3), 1.1(peak 4)；
And calculate the relative peak area of each characteristic peak and S peak, and should be within the scope of the 30% of setting, relative peak area setting is:
0.6(mannose), 16.5(glucose), 1.00(galactose), 0.4(fucose).
Experimental example 2: the assay research of each monosaccharide in Coriolous Dersicolor (Fr.) Quel medical material and preparation thereof
Due to Coriolous Dersicolor (Fr.) Quel medical material and preparation determination of polysaccharide many employings spectrophotography thereof and titration measuring, wherein many with
Glucose is reference substance, and this type of method specificity is poor, and accuracy is low, and quality controllability is poor, and the present invention uses the monosaccharide that it is main
The content of component, controls the quality of herbal polysaccharide, and result of the test shows, under conditions selected good separating effect, the line of each monosaccharide
Property, the methodological study such as stability, repeatability, the response rate all meet the requirement of assay, can be used for controlling herbal polysaccharide
Quality, can contain herbal polysaccharide medical material and the quality of preparation by effectively evaluating.Result of the test is as follows:
1, the preparation of solution
Reference substance solution: accurately weigh standard substance mannose, glucose, galactose, fucose monosaccharide each in right amount, with water-soluble
Solution is configured to suitable monosaccharide standard substance liquid.
Deriving of contrast solution: with experimental example 1 " derivative with reference to solution ".
Prepared by need testing solution: take experimental example 1 " need testing solution ".
2, chromatographic condition and system suitability:
Chromatographic column (AQ C with octadecylsilane chemically bonded silica as filler18, 4.6 × 250mm, 5 μm), with acetonitrile and
Phosphate (weighs K2HPO44.4g, the 800mL that adds water dissolve, and add phosphoric acid and adjust pH to 6.5, and are diluted with water to 1000mL) for flowing
Phase according to the form below carries out gradient elution；Column temperature: 35 DEG C；Detection wavelength: 245nm；Sample size: 10 μ l；Flow velocity: 1.0ml/min.
3, Method validation
Linear relationship is investigated: with peak area as vertical coordinate, monosaccharide concentration (mg/mL) is abscissa, draws standard curve, knot
Fruit is as follows:
Test shows, in table in concentration range, peak area (A) and concentration C (mg/mL) are all in good linear relationship.
Instrument precision is tested: the monosaccharide peak area RSD% that respectively mainly comprises of result standard reference material solution is respectively as follows:
Result shows that this instrument precision is preferable.
Test liquid stability test: result need testing solution, in 24 hours, respectively mainly comprises the peak area RSD of monosaccharide respectively
For:
Result shows that need testing solution stability is preferable.
Replica test: repeating test in this way, the content respectively mainly comprising monosaccharide is respectively as follows:
Result shows the method repeatability preferably.
Recovery test: the average recovery rate and the RSD% that respectively mainly comprise monosaccharide are respectively as follows:
Result shows that the method response rate is preferable.
Therefore, the assay of monosaccharide during the method can be used for the Chinese medicine containing polysaccharide and preparation thereof.
4, the determination of content limitation
According to the sample of many batches of Coriolus Versicolor P.E. preparations of detection, and consider to produce practical situation, determine that this product contains Coriolous Dersicolor (Fr.) Quel temporarily
Polysaccharide, must not be less than 25% in terms of D-MANNOSE, D-Glucose, galactose, L-fucose total amount.
Specific embodiment is as follows:
Embodiment 1 ultra-performance liquid chromatography measures the monosaccharide of Coriolus Versicolor P.E. preparation polysaccharide
1, ultra-performance liquid chromatography measures the characteristic spectrum of Coriolus Versicolor P.E. preparation: according to high performance liquid chromatography (China
2010 editions one annex VI D of pharmacopeia)；
Chromatographic condition instrument: watersUPLC, DAD detector;Chromatographic column: octadecylsilane chemically bonded silica chromatographic column
(Kinetex C18100A, 4.6 × 150mm, 2.6 μm), (weigh K with acetonitrile and phosphate2HPO44.4g, the 800mL that adds water is molten
Solve, add phosphoric acid and adjust pH to 6.5, and be diluted with water to 1000mL) carry out gradient elution for flowing phase according to the form below；Column temperature: 35 DEG C；Inspection
Survey wavelength: 245nm；Sample size: 2.0 μ l；Flow velocity: 0.9ml/min.
Preparation with reference to solution accurately weighs galactose standard substance monosaccharide in right amount, is configured to suitable monosaccharide with water dissolution
Standard substance liquid.
Derivative with reference to solution measures above-mentioned reference solution in right amount, adds 0.5mLNaOH(0.5M), 0.8mLPMP methanol solution
(0.5mol/L), mixing, in 70 DEG C of derivative reaction 45min, adding hydrochloric acid (0.5mol/L) 0.7mL(PH value after letting cool is 4), and
It is diluted with water to 5mL, 5000rpm and is centrifuged 10min, take supernatant and add the extraction of equal-volume chloroform, vibrate, be centrifuged, discard lower floor's chlorine
Imitative, it is repeated 3 times, takes upper water solution, obtain with reference to solution.
Need testing solution preparation weighs Coriolous Dersicolor (Fr.) Quel preparation 0.5g, and the 5mL that adds water makes dissolving, adds 4 times amount ethanol, shakes up, from
Heart 10min, precipitation separation, and precipitate 3 times with absolute ethanol washing, precipitation adds 8mL water makes dissolving, then (concentration is to add dilute sulfuric acid
10%) 7mL, shakes up, and is heated to reflux hydrolyzing 6h, lets cool, and adjusts pH the most neutral by NaOH solution, and is transferred to 25mL measuring bottle, adds water dilute
Releasing to scale, centrifugal, precision measures supernatant 0.5mL, derives by " derivative with reference to solution " method, obtains need testing solution
Algoscopy precision respectively is drawn with reference to solution and each 2 L of sample solution, injects chromatograph of liquid, measures, records color
Spectrogram, to obtain final product.
Should present 4 characteristic spectrums in test sample characteristic spectrum, the peak corresponding with galactose object of reference peak, for S peak, is counted
Calculate the relative retention time of each characteristic peak and S peak, should be within the scope of the 5% of setting, relative retention time setting is: 0.5
(mannose), 0.9(glucose), 1.0(galactose), 1.1(fucose)；And calculate the relative peak area of each characteristic peak and S peak,
Should be within the scope of the 30% of setting, relative peak area setting be: 0.6(mannose), 16.5(glucose), 1.0(gala
Sugar), 0.4(fucose).The results are shown in Table 1.
Table 1 Coriolus Versicolor P.E. UPLC characteristic spectrum relative retention time and relative peak area
2, ultra-performance liquid chromatography measures the contents of monosaccharides of Coriolus Versicolor P.E. preparation: according to high performance liquid chromatography (China
2010 editions one annex VI D of pharmacopeia).
Chromatographic condition is with " chromatographic condition " under embodiment 1.
Prepared by mixing contrast solution: take D-MANNOSE reference substance, D-Glucose reference substance, galactose reference substance, L-rock algae
Sugar reference substance is appropriate, the most accurately weighed, be dissolved in water and be diluted to every 1ml containing D-MANNOSE 0.5mg, D-Glucose 10mg,
Galactose 0.5mg, the mixed solution of L-fucose 0.3mg, to obtain final product.
Deriving of mixing contrast solution: with " derivative with reference to solution " under embodiment 1.
Prepared by need testing solution: with under embodiment 1 " need testing solution "
Algoscopy precision respectively draws mixing contrast solution and each 2 L of need testing solution, injects superelevation chromatograph of liquid,
Measure, to obtain final product.
The every 1g of this product is containing krestin in terms of mannose, glucose, galactose, fucose total amount, and this product contains krestin
In terms of D-MANNOSE, D-Glucose, galactose, L-fucose total amount, must not be less than 25%.
Table 2 UPLC method measures contents of monosaccharides in Coriolus Versicolor P.E.
The monosaccharide of embodiment 2 high effective liquid chromatography for measuring Coriolus Versicolor P.E. preparation polysaccharide
1, the characteristic spectrum of high effective liquid chromatography for measuring Coriolus Versicolor P.E. preparation: according to high performance liquid chromatography (middle traditional Chinese medicines
2010 editions one annex VI D of allusion quotation)
Chromatographic condition chromatographic condition and system suitability instrument: AgilentHPLC1100, with octadecyl silicon
Alkane bonded silica gel is chromatographic column (the AQ C of filler18, 4.6 × 250mm, 5 μm), (weigh K with acetonitrile and phosphate2HPO4
4.4g, the 800mL that adds water dissolve, and add phosphoric acid and adjust pH to 6.5, and are diluted with water to 1000mL) carry out gradient for flowing phase according to the form below
Eluting；Column temperature: 35 DEG C；Detection wavelength: 245nm；Sample size: 10 μ l；Flow velocity: 1.0ml/min.
Preparation with reference to solution is prepared with " reference solution " under embodiment 1.
Prepared by need testing solution: with implementing under row 1 " need testing solution "
Algoscopy precision respectively is drawn with reference to solution and each 10 L of need testing solution, injects high performance liquid chromatograph, surveys
Fixed, to obtain final product.
Should present 4 characteristic spectrums in test sample characteristic spectrum, the peak corresponding with galactose object of reference peak, for S peak, is counted
Calculate the relative retention time of each characteristic peak and S peak, should be within the scope of the 5% of setting, relative retention time setting is: 0.5
(mannose), 0.9(glucose), 1.0(galactose), 1.1(fucose)；And calculate the relative peak area of each characteristic peak and S peak,
Should be within the scope of the 30% of setting, relative peak area setting be: 0.6(mannose), 16.5(glucose), 1.0(gala
Sugar), 0.4(fucose).
Table 3 Coriolus Versicolor P.E. HPLC characteristic spectrum relative retention time and relative peak area
2, the contents of monosaccharides of high effective liquid chromatography for measuring Coriolus Versicolor P.E. preparation: according to high performance liquid chromatography (middle traditional Chinese medicines
2010 editions one annex VI D of allusion quotation)
Chromatographic condition is with " chromatographic condition " under embodiment 2.
Prepared by mixing contrast solution: with " mixing contrast solution " under embodiment 1.
Prepared by need testing solution: with under embodiment 1 " need testing solution "
Algoscopy precision respectively draws mixing contrast solution and each 10 L of need testing solution, injects high performance liquid chromatograph,
Measure, to obtain final product.
The every 1g of this product is containing krestin in terms of mannose, glucose, galactose, fucose total amount, and this product contains krestin
In terms of D-MANNOSE, D-Glucose, galactose, L-fucose total amount, must not be less than 25%.
Table 4 HPLC method measures contents of monosaccharides in Coriolus Versicolor P.E.
Embodiment 3 ultra-performance liquid chromatography measures the monosaccharide of Coriolous Dersicolor (Fr.) Quel polysaccharide from medicinal materials
1, ultra-performance liquid chromatography measures the characteristic spectrum of Coriolus Versicolor P.E. preparation: according to high performance liquid chromatography (China
2010 editions one annex VI D of pharmacopeia)
Chromatographic condition is with under embodiment 1 " chromatographic condition "
The preparation of reference solution is with " reference solution " under embodiment 1.
Need testing solution preparation weighs Coriolous Dersicolor (Fr.) Quel medical material 5g, and precision adds water 120mL, and weighed weight is heated to reflux 1 hour, puts
Cold, more weighed weight, supply the weight of less loss with water, shake up, centrifugal, precision measures 40ml, is evaporated to do, and add water 5mL
Make dissolving, add 4 times amount dehydrated alcohol, shake up, centrifugal 10min, precipitation separation, and precipitate 3 times with absolute ethanol washing, heavy
Shallow lake adds 8mL water makes dissolving, then adds dilute sulfuric acid (concentration is 10%) 7mL, shakes up, and is heated to reflux hydrolyzing 6h, lets cool, use NaOH solution
Adjusting pH the most neutral, and be transferred to 25mL measuring bottle, be diluted with water to scale, centrifugal, precision measures supernatant 0.5mL, by " with reference to molten
Liquid derivative " method derives, and obtains need testing solution.
Algoscopy precision respectively is drawn with reference to solution and each 2 L of sample solution, injects chromatograph of liquid, measures, records color
Spectrogram, to obtain final product.
Should present 4 characteristic spectrums in test sample characteristic spectrum, the peak corresponding with galactose object of reference peak, for S peak, is counted
Calculate the relative retention time of each characteristic peak and S peak, should be within the scope of the 5% of setting, relative retention time setting is: 0.5
(mannose), 0.9(glucose), 1.0(galactose), 1.1(fucose)；And calculate the relative peak area of each characteristic peak and S peak,
Should be within the scope of the 30% of setting, relative peak area setting be: 0.8(mannose), 22.5(glucose), 1.0(gala
Sugar), 0.3(fucose).
Table 5 Coriolous Dersicolor (Fr.) Quel medical material UPLC characteristic spectrum relative retention time and relative peak area
2, the contents of monosaccharides of ultra-performance liquid chromatography mensuration Coriolous Dersicolor (Fr.) Quel polysaccharide from medicinal materials hydrolysis: photograph high performance liquid chromatography (in
2010 editions one annex VI D of state's pharmacopeia).
Chromatographic condition is with " chromatographic condition " under embodiment 1.
Prepared by mixing contrast solution: with " mixing contrast solution " under embodiment 1.
Prepared by need testing solution: with implementing under row 3 " need testing solution ".
Algoscopy precision respectively draws mixing contrast solution and each 2 L of need testing solution, injects superelevation chromatograph of liquid,
Measure, to obtain final product.
The every 1g of this product is containing krestin in terms of mannose, glucose, galactose, fucose total amount, and this product contains krestin
In terms of D-MANNOSE, D-Glucose, galactose, L-fucose total amount, must not be less than 3%.
Table 6 UPLC method measures contents of monosaccharides in Coriolous Dersicolor (Fr.) Quel medical material
The monosaccharide of embodiment 4 high effective liquid chromatography for measuring Coriolous Dersicolor (Fr.) Quel polysaccharide from medicinal materials
1, the characteristic spectrum of high effective liquid chromatography for measuring Coriolous Dersicolor (Fr.) Quel medical material: according to high performance liquid chromatography (Chinese Pharmacopoeia 2010
One annex VI D of version)
Chromatographic condition chromatographic condition and system suitability instrument: AgilentHPLC1100, with octadecyl silicon
Alkane bonded silica gel is chromatographic column (the AQ C of filler18, 4.6 × 250mm, 5 μm), (weigh K with acetonitrile and phosphate2HPO4
4.4g, the 800mL that adds water dissolve, and add phosphoric acid and adjust pH to 6.5, and are diluted with water to 1000mL) carry out gradient for flowing phase according to the form below
Eluting；Column temperature: 35 DEG C；Detection wavelength: 245nm；Sample size: 10 μ l；Flow velocity: 1.0ml/min.
Preparation with reference to solution is prepared with " reference solution " under embodiment 1.
Prepared by need testing solution: with implementing under row 3 " need testing solution "
Algoscopy precision respectively is drawn with reference to solution and each 10 L of need testing solution, injects high performance liquid chromatograph, surveys
Fixed, to obtain final product.
Should present 4 characteristic spectrums in test sample characteristic spectrum, the peak corresponding with galactose object of reference peak, for S peak, is counted
Calculate the relative retention time of each characteristic peak and S peak, should be within the scope of the 5% of setting, relative retention time setting is:
0.45(mannose), 0.90(glucose), 1.00(galactose), 1.16(fucose)；And it is relative with S peak to calculate each characteristic peak
Peak area, should setting ± 30% within the scope of, relative peak area setting is: 0.8(mannose), 22.5(glucose),
1.0(galactose), 0.3(fucose).
Table 7 Coriolous Dersicolor (Fr.) Quel medical material HPLC characteristic spectrum relative retention time and relative peak area
2, the contents of monosaccharides of high effective liquid chromatography for measuring Coriolous Dersicolor (Fr.) Quel medical material: according to high performance liquid chromatography (Chinese Pharmacopoeia 2010
One annex VI D of version)
Chromatographic condition is with " chromatographic condition " under embodiment 2.
Prepared by mixing contrast solution: with " mixing contrast solution " under embodiment 1.
Prepared by need testing solution: with under embodiment 3 " need testing solution "
Algoscopy precision respectively draws mixing contrast solution and each 10 L of need testing solution, injects high performance liquid chromatograph,
Measure, to obtain final product.
The every 1g of this product is containing krestin in terms of mannose, glucose, galactose, fucose total amount, and this product contains krestin
In terms of D-MANNOSE, D-Glucose, galactose, L-fucose total amount, must not be less than 3%.
Table 8 HPLC method measures contents of monosaccharides in Coriolous Dersicolor (Fr.) Quel medical material
Claims (2)
1. a Coriolous Dersicolor (Fr.) Quel and the detection method of the preparation containing Coriolous Dersicolor (Fr.) Quel, it is characterised in that Coriolous Dersicolor (Fr.) Quel and the preparation containing Coriolous Dersicolor (Fr.) Quel use alcohol
Heavy extraction crude polysaccharides, becomes monosaccharide fragments with acid hydrolysis polysaccharide, then monosaccharide derivatization reaction is made need testing solution, use ultra high efficiency
The characteristic spectrum of the monosaccharide fragments of mannose, glucose, galactose and fucose in liquid chromatography detection need testing solution
And the content of each monosaccharide, set up characteristic spectrum；
The preparation method of described need testing solution is:
Taking Coriolous Dersicolor (Fr.) Quel medical material 3～6g, add water appropriate, heating and refluxing extraction, extracting solution is centrifuged, measures appropriate extracting solution, concentrating under reduced pressure
To in right amount, or take Coriolous Dersicolor (Fr.) Quel preparation 0.5～1g, add water and make dissolving；Add the dehydrated alcohol of 3～6 times of volumes, divide and take precipitation, precipitation
Being dissolved in water, and add the dilute sulfuric acid of weight content 10%, 100-120 DEG C is heated to reflux hydrolyzing 5～6 hours, and after hydrolysis, solution is adjusted
PH the most neutral also constant volume, Aspirate supernatant, put in tool plug test tube, and the NaOH solution of addition 0.5mol/L and the methanol of PMP are molten
Liquid, adjusts pH to neutral and constant volume after 60～80 DEG C of derivative reaction 30-60min, reaction completely, takes supernatant and add equal-volume three
Chloromethanes shaking is extracted, and discards organic solution, repetitive operation 3 times, and aqueous solution is evaporated to use acetonitrile constant volume in right amount, as confession
Test sample solution；
The method for building up of described characteristic spectrum is: take D-MANNOSE, D-Glucose, galactose and L-fucose reference substance suitable
Amount, is dissolved in water, and by above-mentioned need testing solution preparation mixing reference substance solution, precision draws mixing reference substance solution and confession respectively
Test sample solution, injects chromatograph of liquid, measures each contents of monosaccharides；The characteristic spectrum of need testing solution should present mannose, Portugal
Grape sugar, galactose, 4 characteristic peaks of fucose, it should be consistent with the retention time at corresponding object of reference peak, wherein to appoint respectively
Corresponding peak, object of reference peak of anticipating is S peak, calculates each characteristic peak and the relative retention time at S peak, relative peak area；
The relative retention time of the described characteristic peak by each monosaccharide is or/and peak area and content control and evaluates Coriolous Dersicolor (Fr.) Quel
The Quality Process of medical material and preparation thereof is:
(1) Coriolus Versicolor P.E. real and fake discrimination:
The peak corresponding with galactose object of reference peak, for S peak, calculates the relative retention time of each characteristic peak and S peak；
(2) Coriolous Dersicolor (Fr.) Quel medical material real and fake discrimination:
The peak corresponding with galactose object of reference peak, for S peak, calculates the relative retention time of each characteristic peak and S peak；
Each characteristic peak exists, and relative retention time and relative peak area meet the requirements, and evaluates Coriolous Dersicolor (Fr.) Quel and the system containing Coriolous Dersicolor (Fr.) Quel with this
The true and false of agent；
Described Ultra Performance Liquid Chromatography testing conditions: with octadecylsilane chemically bonded silica as filler, flow velocity is 0.3 1 1.
0 ml/min；Detection wavelength 245nm；With acetonitrile as mobile phase A, with phosphate buffered saline(PBS) as Mobile phase B, eluent gradient
Eluting；
Described phosphate buffered saline(PBS) collocation method: weigh K2HPO44.4g, the 800mL that adds water dissolve, and add phosphoric acid and adjust pH extremely
6.5, and it is diluted with water to 1000mL.
Coriolous Dersicolor (Fr.) Quel the most according to claim 1 and the detection method of the preparation containing Coriolous Dersicolor (Fr.) Quel, it is characterised in that the method is at cloud
Sesame medical material, containing the preparation of Coriolous Dersicolor (Fr.) Quel, including answering of Coriolous Dersicolor (Fr.) Quel single medicinal substances extract, Coriolous Dersicolor (Fr.) Quel effective site or Coriolous Dersicolor (Fr.) Quel effective part group
Application in terms of the quality control of square preparation and evaluation.
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