CN106706774B - The method that area normalization method measures crocin constituents in west safflower medicinal material - Google Patents
The method that area normalization method measures crocin constituents in west safflower medicinal material Download PDFInfo
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Abstract
The invention discloses the methods of crocin constituents in a kind of area normalization method measurement west safflower medicinal material, establish west safflower quality of medicinal material control method, isolate 12 main crocin constituents, with relatively stable, inexpensive crocin-I for internal reference object, assay is carried out to the total glycosides of west safflower in west safflower medicinal material using area normalization method, solves the problems, such as when HPLC method measurement west safflower medicinal material that multiple crocin reference substances are not easy to obtain when the separation of multiple crocin constituents and assay.With crocin-I for internal reference object, the relative correction factor of-I pair of crocin-II of crocin is established, the content of crocin-II is calculated using relative correction factor；And be compared with crocin-I in 32 batches of west safflowers of the external standard method of pharmacopeia use and II content, survey the feasibilities for commenting method to verify one more.Solve the problems, such as that-II reference substance property of crocin is unstable, expensive in pharmacopeia measuring method.
Description
Technical field
The present invention relates to active ingredient of Chinese herbs analysis technical fields, specifically, being to be related to a survey to comment method to return with area more
The method of crocin constituents in the measurement west safflower medicinal material that one change method combines.
Background technique
West safflower (Crocus sativus L.) is also known as safflower, is only used as medicine with the red column cap in three, gynoecium top, manually
100000-150000 flower of picking could obtain one kilogram of safflower dry product, therefore expensive, be rare traditional Chinese medicine, have
The effect of activating microcirculation and removing stasis medicinal, removing pattogenic heat from the blood and toxic material from the body, resolving stagnation for tranquilization.Principle active component is crocin (crocins), is a kind of red
Water soluble carotenoid class compound is a series of ester glycosides that crocetin and glucose or gentiobiose are combined into, mesh
It is preceding separated to obtain the crocin that aglycon is 16 kinds of crocetin different cis-trans-isomers.Separate sources west all over the world
It is secondly crocin-II, the cultivation of the crocin place of production, dry and storage etc. with-I content highest of crocin in safflower
It is very big on the influence of crocin content, therefore saffron glucoside appropriate is required in terms of west safflower production and processing and quality control
Content assaying method, " Chinese Pharmacopoeia " version in 2015 in crocin crocin-I and glycosides-II be index to west safflower
Quality control, it is desirable that the two total content is not less than 10%, but crocin-I and glycosides-II only account for each west safflower in certain samples
70% or so of total glycosides, and as degradation or isomerization can occur for the increase of storage time, trans- west safflower-I and glycosides-II,
Part switchs to other cis-isomers, and after crocin oral administration, all crocins can be hydrolyzed to aglycon west safflower
Acid is absorbed into blood action, therefore influence of the height of the total glycosides content of west safflower to drug effect is bigger, and " Chinese Pharmacopoeia " is according to western red
The total content of flower glycosides-I and glycosides-II is standard, cannot reflect west safflower quality of medicinal material comprehensively；West safflower is mainly used as in the world
Pigment and fragrance, as the international standard ISO3632-2 (2010 editions) of food additives, with spectrophotometry in λ max 440nm
Measure west safflower aqueous extract color value come carry out quality control with classification, it is specified that crocin color value be greater than 190 be primes,
This method can not accurate quantification crocin content.Therefore, " Chinese Pharmacopoeia " and international standard ISO 3632-2 are to safflower
The standard of quality evaluation be both needed to it is further perfect, need to establish be suitble to production and processing and quality control the total glycosides of west safflower containing measure
Determine method.
It is generally acknowledged that spectrophotometry characteristic is not strong, but west safflower is medicinal material very special simply, and effective component collects
Middle Yu Zhutou, content is high and relatively easy, mainly only three constituents of saffron glucoside, safranal and picrocrocin, in visible light
Only have crocins to have absorption at area 440nm, other compositions are noiseless, measure total west with visible spectrophotometry (440nm)
The method of carthamic acid content, method is easy, easy to spread, but this method is only suitable for production and processing person's use, if having artificial adulterated
Equal behaviors are then not suitable for using.
Summary of the invention
The technical problem to be solved by the present invention is to only measure crocin-I and crocin-II for " Chinese Pharmacopoeia "
Content, cannot embody the real content of total crocin comprehensively, and total crocin color value of " international standard " measurement is not energetic
The shortcomings that content of total crocin.The present invention surveys two kinds of fixed crocin contents of more evaluation and tests and area normalization method survey for one
Fixed total crocin content combines, and realizes the assay that a reference substance completes each crocin constituents, reduces
Testing cost, and can thoroughly evaluating west safflower quality, for west safflower evaluation of medical materials' quality method it is perfect provide it is more preferable
Technical Reference.
The present invention uses high-efficient liquid phase technique, western red in the technologies measurement medicinal material such as wavelength switching by preferred chromatographic condition
Flower glycosides separates major part crocin constituents, but the aglycon of crocin is made of seven conjugated double bonds, oxidizable shakiness
It is fixed, therefore crocin reference substance is expensive, all crocins component contents of accurate quantification, testing cost is very high
Expensive, the present invention, which is used, carries out assay to the total glycosides of west safflower in west safflower medicinal material using area normalization method (ANM), solves
Multiple crocin reference substances are not easy the problem of obtaining when HPLC method assay.
The present invention with crocin-I for internal reference object, establish the relative correction of-I pair of crocin-II of crocin because
Son calculates the content of crocin-II using relative correction factor (one surveys comment more)；And the external standard method used with pharmacopeia
Crocin-I and-II content of glycosides are compared in 32 batches of west safflowers, survey the feasibilities for commenting method to verify one more.As a result west safflower
Glycosides-I is reproducible with respect to the relative correction factor of crocin-II, and the one of crocin-II surveys the meters for commenting method more in west safflower
Calculation value and pharmacopeia external standard method result are almost the same, solve in pharmacopeia measuring method-II reference substance property of crocin not
Stable, expensive problem.
In order to solve the above technical problems, the present invention provides west safflowers in a kind of area normalization method measurement west safflower medicinal material
The method of methods of glycosides, comprising steps of
A. chromatographic condition is determined；
B. reference substance solution is prepared:
Mixed reference substance solution: precision weighs-II reference substance of-I reference substance of crocin and crocin, is placed in brown amount
In bottle, scale is dissolved and be settled to Diluted Alcohol, obtains mixed reference substance solution, is shaken up, it is spare；
- I reference substance solution of crocin: precision weighs-I reference substance of crocin and sets in brown measuring bottle, molten with Diluted Alcohol
Scale is solved and be settled to ,-I reference substance solution of crocin is obtained；
C. prepare test solution: referring under Chinese Pharmacopoeia version west safflower in 2015, precision weighs west safflower sample powder
End is placed in brown measuring bottle, adds Diluted Alcohol 40mL, ice-bath ultrasonic process 20min, is placed room temperature, is added ethyl alcohol to be settled to scale, shake
Even, filtering with microporous membrane takes subsequent filtrate, obtains test solution；
D. according to the content of the quantified by external standard method crocin-I of version Chinese Pharmacopoeia in 2015 and glycosides-II；
E. one the contents for commenting method QMSA measurement crocin-I and glycosides-II are surveyed more；F. determined by ultraviolet spectrophotometry is always western
The content of carthamic acid；
G. area normalization method measures the content of total crocin.
Preferably, in the step a, chromatographic condition be Ultimate XB C18 chromatographic column, chromatographic column be 250mm ×
4.6mm,5μm；Column temperature: 30 DEG C；Mobile phase: water-methanol, gradient elution, 0-60min, methanol: 20%-100%；DAD detector
Wave-length coverage 200-600nm, Detection wavelength 440nm, flow velocity: 1.0mLmin-1；Sample volume: 10 μ L.
Preferably, in the step c, the ice-bath ultrasonic is that 250W, 40KHz handle 20min.
Preferably, in the step c, the aperture of miillpore filter is 0.22 μm.
Preferably, the step d, further to take the mixed reference substance solution of crocin-I and glycosides-II in step b
1,2,4,8,10,20 μ L, by chromatographic condition in step a, sample introduction is analyzed, peak area is measured, respectively with crocin-I and glycosides-II
Mass concentration (X, μ g) carries out linear regression to its corresponding peak area (Y), obtains regression equation, precision measure in step c for examination
10 μ L of product solution, injects hplc determination, and corresponding peak area substitutes into regression equation calculation glycosides-I and-II content of glycosides.
Preferably, the step e, further for, take mixed reference substance solution in step b, respectively sample introduction 1,2,4,8,10,
15,20 μ L records peak area, using Supplements method, with crocin-I for internal reference object, calculates-I pair of west safflower of crocin
The relative correction factor f of glycosides-IIk/s, fk/sCalculation formula: fk/s=(Cs×Ak)/(Ck×As), ingredient (crocin-to be measured
II) mass concentration calculation formula: Ck′=(Cs × Ak′)/(fk/s× As),
Wherein Cs is internal reference object (crocin-I) mass concentration, and As is that internal reference looks for spectral peak peak area, CkFor west safflower
- II reference substance mass concentration of glycosides, AkFor-II reference substance chromatographic peak peak area of crocin, Ck′For west safflower in west safflower sample
- II mass concentration of glycosides, Ak ' are-II chromatographic peak peak area of crocin in west safflower sample.
Preferably, the step f, further for, precision draw-I reference substance solution 0.1 of crocin, 0.2,0.4,
0.6,0.8,1.0mL is placed in 10mL measuring bottle, is diluted to scale with Diluted Alcohol, makees blank control with Diluted Alcohol, using it is ultraviolet-can
See spectrophotometry, absorbance is measured at 440nm wavelength, obtains standard curve；Test solution in accurate aspiration step c
5mL, sets in 50mL brown measuring bottle, is diluted to scale with Diluted Alcohol, and extinction is measured at 440nm wavelength with visible spectrophotometry
Degree, is reference standards with crocin-I, calculates total crocin content by standard curve.
Preferably, the step g, further for, weigh west safflower sample powder about 10mg, it is accurately weighed, set 50mL palm fibre
In colo(u)r specification bottle, add Diluted Alcohol 40mL, ice-bath ultrasonic (250W, 40KHz) handles 20min, places room temperature, ethyl alcohol is added to be settled to quarter
Degree, shakes up, and 0.22 μm of miillpore filter filtration, precision draws each 10 μ L of reference substance solution in subsequent filtrate and step b, injects liquid phase color
Spectrometer measurement, minute are no less than 60 minutes, are analyzed by mass spectrometry simultaneously, Mass Spectrometry Conditions are as follows: ion source: atmosphere piezo jet
Mist ion source (ESI)；Detection pattern: negative ions；Full scan karyoplasmic ratio (m/z): 50-1200；Dry temperature degree: it 350 DEG C, does
Pathogenic dryness flow: 12.0Lmin-1, atomization gas pressure: 35.0psi；Capillary voltage: 4.5kV；Collision voltage 1.0V；Into matter
The flow rate of mobile phase of spectrum branches to 0.4mLmin-1.It is control with crocin-I, by CTotal glycosides=(CGlycosides-I×ATotal glycosides)/AGlycosides-IIt calculates
The content of total crocin,
Wherein AGlycosides-IFor-I chromatographic peak peak area of crocin in sample；ATotal glycosidesIt is western red for west safflower sample 440nm lower 12
The sum of flower glycoside chromatographic peak peak area；CGlycosides-IFor the mass concentration of crocin-I in the west safflower sample of external standard method；CTotal glycosides
For crocin mass concentration total in west safflower sample.
Compared with prior art, of the present invention one the measurement west safflowers for commenting method to combine with area normalization method are surveyed more
The method of crocin constituents in medicinal material achieving the following effects:
The application with crocin-I for internal reference object, while being detected as defined in " Chinese Pharmacopoeia " using " one surveys comment more " method
The content of two kinds of crocins is compared with Chinese Pharmacopoeia method, to reduce expensive-II reference substance of crocin
It uses.And similar according to each crocin structure (is different cis-trans isomerisms of the crocetin in conjunction with different number glucose
Body), maximal ultraviolet absorption is identical, while being control with crocin-I, calculates total crocin using area normalization method
Content, and it is compared analysis with the content of the total crocin of spectrophotometry under established 440nm seminar's early period,
Establishing can the method for detecting total crocin content synchronous with crocin-I and glycosides-II.It, will compared to " Chinese Pharmacopoeia " method
One surveys the total crocin contents for evaluating and testing fixed two kinds of crocin contents and area normalization method measurement more combines, and realizes
One reference substance completes the assay of crocin, testing cost, and the quality of energy thoroughly evaluating west safflower is reduced, for west
The perfect of flos carthami quality evaluating method provides superior technique reference.
Containing 16 kinds of different saffron glucosides in safflower, wherein crocin-I and-II content of crocin account for 70%
More than, after oral administration, each crocin plays drug effect, but " middle traditional Chinese medicines with the aglycon crocetin absorbed into serum for removing glycosyl
Allusion quotation " 2015 editions only measure crocin-I and-II content of crocin, cannot embody the content of total crocin, and west safflower
In the world without medicinal standard, only as the quality standard ISO 3632-2 of food additives, west is measured with spectrophotometry
The color value of safflower aqueous solution 440nm it is carried out quality control with classification, it is specified that crocin color value be greater than 190 be I grade of product,
150-190 is II grade of product, and 100-150 is III grade of product, but measurement color value cannot provide the content of total crocin.To the west of the present invention
Carthamic acid-I is internal reference object, establishes the relative correction factor of-I pair of crocin-II of crocin, utilizes relative correction factor meter
The content of crocin-II is calculated, realizes that a survey more and comments (QMSA)；And simultaneously with crocin-I to compare, peak area normalization
(ANM) method detects the content of total crocin in west safflower.As a result the calculated value of the QMSA method of crocin-II and pharmacopeia is outer
Mark method measurement result is almost the same.Total crocin content that total crocin content that ANM is calculated is measured with UV method is without significant
Sex differernce.The assay that a reference substance completes crocin is realized, reduces testing cost, and energy thoroughly evaluating is western red
Colored quality.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes a part of the invention, this hair
Bright illustrative embodiments and their description are used to explain the present invention, and are not constituted improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is that HPLC schemes under mixing reference substance (A) 440nm wavelength；Wherein: 1- crocin-I；2- crocin-II；
Fig. 2 is that HPLC schemes under sample 440nm wavelength；Wherein: 1- crocin-I；2- crocin-II；3- west safflower
Glycosides-III；4- crocin-IV；
Fig. 3 is that sample wavelength switches HPLC figure；
Fig. 4 is sample mass spectrum total ion current figure.
Specific embodiment
As used some vocabulary to censure specific components in the specification and claims.Those skilled in the art answer
It is understood that hardware manufacturer may call the same component with different nouns.This specification and claims are not with name
The difference of title is as the mode for distinguishing component, but with the difference of component functionally as the criterion of differentiation.Such as logical
The "comprising" of piece specification and claim mentioned in is an open language, therefore should be construed to " include but do not limit
In "." substantially " refer within the acceptable error range, those skilled in the art can within a certain error range solve described in
Technical problem basically reaches the technical effect.Specification subsequent descriptions are to implement better embodiment of the invention, so described
Description is the range that is not intended to limit the invention for the purpose of illustrating rule of the invention.Protection scope of the present invention
As defined by the appended claims.
Below in conjunction with attached drawing, invention is further described in detail, but not as a limitation of the invention.
Embodiment 1:
A. chromatographic condition is determined
Ultimate XB C18 chromatographic column (250mm × 4.6mm, 5 μm)；Column temperature: 30 DEG C；Mobile phase: water (A)-methanol
(B), gradient elution, 0-60min, B:20%-100%；DAD wave-length coverage 200-600nm, Detection wavelength 440nm, flow velocity:
1.0mL·min-1；Sample volume: 10 μ L.Under the chromatographic condition, each chromatographic peak has preferable baseline separation；
B. reference substance solution is prepared
Precision weighs-I reference substance 5.96mg of crocin, and-II reference substance 2.96mg of crocin sets 100mL brown amount
In bottle, is dissolved with Diluted Alcohol and be settled to scale.Obtaining concentration is 59.6 μ gmL-1With 29.6 μ gmL-1Mixing reference substance it is molten
Liquid shakes up, spare.
- I reference substance solution of crocin: precision weighs-I reference substance of crocin and sets in brown measuring bottle, molten with Diluted Alcohol
Scale is solved and be settled to ,-I reference substance solution of crocin is obtained；
C. test solution is prepared
Referring under Chinese Pharmacopoeia version west safflower in 2015, west safflower sample powder about 10mg is weighed, it is accurately weighed, it sets
In 50mL brown measuring bottle, add Diluted Alcohol 40mL, ice-bath ultrasonic (250W, 40KHz) handles 20min, places room temperature, adds ethyl alcohol constant volume
To scale, shake up, the filtration of 0.22 μm of miillpore filter, take subsequent filtrate to get.
D.2015 the quantitative crocin-of year version " Chinese Pharmacopoeia " external standard method (external standard method, ESM)
I and glycosides-II content
1,2,4,8,10,20 μ L of crocin-I and-II mixed reference substance solution of glycosides in step b is taken, by A chromatographic conditions
Sample introduction is analyzed, measures peak area.Respectively with crocin-I and-II mass concentration of glycosides (X, μ g) to its corresponding peak area (Y) into
Row linear regression obtains the regression equation Y=941.16X-4.0829 (r=0.9999) and Y=of glycosides-I and glycosides-II respectively
1108.9X-0.8237 (r=0.9999).The result shows that crocin-I and glycosides-II respectively in 0.0596-1.192 μ g and
Respectively peak area product is in good linear relationship to 0.0296-0.592 μ g with its.10 μ L of test solution in accurate aspiration step c, note
Enter hplc determination, corresponding peak area substitutes into regression equation calculation glycosides-I and-II content of glycosides.
E. one the contents for commenting method (QMSA) measurement crocin-I and glycosides-II are surveyed more
Using Supplements method, with crocin-I for internal reference object, the opposite of-I pair of crocin-II of crocin is calculated
Correction factor fk/s measures the content of two crocins-I and crocin-II with standard items crocin-I.It takes
Lower mixed reference substance solution in step b, 1,2,4,8,10,15,20 μ L of sample introduction, records peak area respectively, using Supplements method,
With crocin-I for internal reference object, the relative correction factor f of-I pair of crocin-II of crocin is calculatedk/s, fk/sIt calculates public
Formula: fk/s=(Cs×Ak)/(Ck×As).Ingredient (crocin-II) mass concentration calculation formula to be measured: Ck′=(Cs × Ak′)/
(fk/s×As).Cs is internal reference object (crocin-I) mass concentration in two formulas, and As is that internal reference looks for spectral peak peak area, CkFor west
- II reference substance mass concentration of carthamic acid, AkFor-II reference substance chromatographic peak peak area of crocin, Ck′For west safflower sample Chinese and Western
- II mass concentration of carthamic acid, Ak′For-II chromatographic peak peak area of crocin in west safflower sample.
F.UV method measures the content of total crocin
Test solution 5mL under in accurate aspiration step c, sets in 50mL brown measuring bottle, is diluted to scale with Diluted Alcohol,
West safflower medicinal material absorbance is measured at 440nm wavelength with visible spectrophotometry, is reference standards with crocin-I, is pressed
Standard curve calculates separately total crocin content.
G. area normalization method measures the content of total crocin
West safflower sample powder about 10mg is weighed, it is accurately weighed, it sets in 50mL brown measuring bottle, adds Diluted Alcohol 40mL, ice bath
Ultrasonic (250W, 40KHz) handles 20min, places room temperature, adds ethyl alcohol to be settled to scale, shake up, 0.22 μm of miillpore filter filtration,
Precision draws each 10 μ L of reference substance solution in subsequent filtrate and step b, hplc determination is injected, due to western red in west safflower
Flower glycosides, picrocrocin and safranal three classes main compound have characteristic absorption at 440nm, 250nm and 330nm, but absorb
Intensity differs greatly, so using wavelength handoff technique.By above-mentioned experiment condition, take reference substance and west safflower sample solution into
Sample analysis, obtains reference substance and west safflower sample high-efficient liquid phase chromatogram and mass spectrum total ion chromatogram.It is isolated at 440nm
12 different crocin constituents are identified, separately have detection of the crocin constituents lower than HPLC method in 4 to limit.To the west of it is red
Flower glycosides-I is control, by CTotal glycosides=(CGlycosides-I×ATotal glycosides)/AGlycosides-ICalculate the content of total crocin.A in formulaGlycosides-IFor west safflower in sample
- I chromatographic peak peak area of glycosides；ATotal glycosidesFor the sum of all chromatographic peak peak areas under west safflower sample 440nm；CGlycosides-IFor external standard method
The mass concentration of crocin-I in west safflower sample；CTotal glycosidesFor crocin mass concentration total in west safflower sample.
Embodiment 2: the measurement of crocin constituents in domestic and international 32 batches of west safflower medicinal materials
1. instrument
1200 high performance liquid chromatograph of Agilent (Agilent company, the U.S.)；6330 ion trap mass spectrometer of Agilent,
Equipped with electric spray ion source (ESI) (Agilent company, the U.S.)；The 45 spectrophotometry instrument (U.S. Lambda
PerkinElmer company).
Reagent and test sample
Standard items: crocin-I (lot number: 111588-201202), crocin-II (lot number: 111589-201304)
It is purchased from National Institute for Food and Drugs Control；Methanol and formic acid are chromatographically pure (Tedia company, the U.S.), remaining chemical reagent
It is that analysis is pure.
32 batches of west safflower medicinal materials are provided by all west safflower Specialty Co-operative Organizations of Jiande City, Zhejiang Province three and different planting bases, warp
Zhejiang University of Traditional Chinese Medicine professor Chen Xilin is accredited as the drying column cap of safron (Crocus Sativus L.).
3. the preparation of reference substance solution
Precision weighs-I reference substance 6.20mg of crocin, sets in 25mL brown measuring bottle, is dissolved and be settled to Diluted Alcohol
Scale.Therefrom precision pipettes 4.0mL and sets in 10mL measuring bottle, and Diluted Alcohol is diluted to scale, shakes up.Obtaining concentration is 99.2 μ gmL-1
Reference substance solution.
4. prepared by test solution
Referring under version " Chinese Pharmacopoeia " west safflower in 2010, west safflower sample powder about 10mg is weighed, it is accurately weighed, it sets
In 50mL brown measuring bottle, add Diluted Alcohol 40mL, ice-bath ultrasonic process 20min, place room temperature, add ethyl alcohol to be settled to scale, shake up,
0.22 μm of miillpore filter filtration, takes subsequent filtrate, and precision draws 5mL, sets in 50mL brown measuring bottle, be diluted to scale with Diluted Alcohol.
5.HPLC chromatographic condition
Ultimate XB C18 chromatographic column (250mm × 4.6mm, 5 μm)；Column temperature: 30 DEG C；Mobile phase: water (A)-methanol
(B), gradient elution, 0-60min, B:20%-100%；DAD 200~600nm of wave-length coverage, Detection wavelength 440nm, flow velocity:
1.0mL·min-1；Sample volume: 10 μ L.Under the chromatographic condition, each chromatographic peak has preferable baseline separation.
6. measuring the selection of wavelength
Reference substance solution and test solution are respectively at 200-700nm progress length scanning, and discovery is both at 440nm
There is absorption maximum, therefore determines that Detection wavelength is 440nm.It is consistent with the maximum absorption wavelength of crocin constituents.
7.UV method standard curve and linear relationship are investigated
Accurate reference substance solution 0.1,0.2,0.4,0.6,0.8,1.0mL of drawing is placed in 10mL measuring bottle respectively, with dilute second
Alcohol is diluted to scale, makees blank control with Diluted Alcohol, and according to UV-VIS spectrophotometry, extinction is measured at 440nm wavelength
Degree, obtaining regression equation is y=0.1130x+0.0012 (r=0.9999).The result shows that crocin-I is in 0.992-9.92 μ
With absorbance in good linear relationship in gmL-1 concentration range.
8.UV method measures total crocin content in sample
Take 32 batches of west safflower medicinal materials, respectively by test solution is prepared under step 4, make blank control with Diluted Alcohol, according to purple
Outside-visible spectrophotometry measures absorbance at 440nm wavelength, calculates separately total crocin content by standard curve, surveys
Surely it the results are shown in Table 1.
9. " Chinese Pharmacopoeia " method measures crocin-I and-II content of crocin
Take 32 batches of west safflower medicinal materials, respectively by preparing test solution under step 4, by " Chinese Pharmacopoeia " chromatographic condition into
Sample.The measurement result of-II total content of crocin-I and crocin is shown in Table 1 in 32 batches of west safflower samples.
10. one surveys the contents for commenting method (QMSA) measurement crocin-I and glycosides-II more
Reference substance solution under step 3 is taken, respectively 1,2,4,8,10,15,20 μ L of sample introduction, peak area is recorded, using multiple spot school
It executes, with crocin-I for internal reference object, calculates the relative correction factor fk/s, fk/s of-I pair of crocin-II of crocin
Calculation formula: fk/s=(Cs × Ak)/(Ck × As).Ingredient (crocin-II) mass concentration calculation formula to be measured: Ck '=
(Cs×Ak′)/(fk/s×As).Cs is internal reference object (crocin-I) mass concentration in two formulas, and As is that internal reference looks for spectral peak peak
Area, Ck are-II reference substance mass concentration of crocin, and Ak is-II reference substance chromatographic peak peak area of crocin, and Ck ' is west
- II mass concentration of crocin in safflower sample, Ak ' are-II chromatographic peak peak area of crocin in west safflower sample.
32 batches of west safflower medicinal materials are taken, respectively by test solution is prepared under step 4, by the lower chromatographic condition sample introduction of step 5.32
The measurement result for criticizing-II total content of crocin-I and crocin in west safflower sample is shown in Table 1.
11. the content that area normalization method measures total crocin
32 batches of west safflower medicinal materials are taken, respectively by test solution is prepared under step 4, by the lower chromatographic condition sample introduction of step 5.With
Crocin-I is control, and the content of total crocin is calculated by the total glycosides of C=(the total glycosides of-I × A of C glycosides)/A glycosides-I.A glycosides-I in formula
For-I chromatographic peak peak area of crocin in sample；The total glycosides of A is the sum of all chromatographic peak peak areas under west safflower sample 440nm；C
Glycosides-I is the mass concentration of crocin-I in the west safflower sample of external standard method；The total glycosides of C is total western red in west safflower sample
Flower glycosides mass concentration.The measurement result of the total glycosides content of west safflower is shown in Table 1 in 32 batches of west safflower samples.
Two kinds of crocins component contents and ANM and UV measurement are total western red in the west safflower that table 1 ESM and QAMS is measured
Flower glycosides content results (%, n=3)
Crocin enters with the action of aglycon crocetin after blood, thus to the west of total its quality of glycosides Content evaluation of flos carthami more
Accurately.Chinese Pharmacopoeia (2015 editions) using HPLC method measurement crocin-I and-II content of glycosides, are not surveyed other methods of glycosides areas
Normalization method content is the consideration for being not easy to obtain and minute cannot be too long based on reference substance；The present invention uses area normalization
Total crocin content of change method measurement is only needed using a kind of reference substance of crocin-I, and minute has 11 in 60min
Crocin constituents are separated, 32 batches of domestic and international west safflower sample measurement results, area normalization method and the UV delivered
There was no significant difference for total crocin content of method measurement, illustrates that area normalization method can be used for the survey of total crocin content
It is fixed.Sample is the dry processing of same producer after the purchase fresh west safflower filigree in the place of production, abroad in the application China
It produces for famous major company, is free of adulterated product through HPLC analysis, therefore easier using the measurement of UV method, but for that may mix
False pain product must then use HPLC to measure the total glycosides of west safflower using area normalization method.
Chinese Pharmacopoeia (2015 editions) are existing statutory standards, it is desirable that the total amount of crocin-I and glycosides-II is greater than 10% and is
Qualified product, but-II standard items of crocin are more expensive, and the aglycon of the two is crocetin, is apo- carotenoids class
Object is closed, the functional group for generating UV absorption is identical, has absorption maximum in 440nm, surveys for one and comments the foundation of method to provide most more
Advantageous endogenous basis.It is the relative correction factor for the crocin-II that different instruments, different chromatographic columns measure in the application, opposite
The RSD of retention time is respectively less than 5%, meets a survey and comments method to require more.Using the crocin-II of Chinese Pharmacopoeia external standard method
Content and the contents for more method being commented to be calculated by a survey are almost the same, with crocin-I for internal reference object, the opposite school of glycosides-II
Positive divisor is 1.18, there is preferable reproducibility and stability, therefore surveys method measurement crocin-I and the glycosides-II commented using one more
Total amount can be used for differentiating whether west safflower medicinal material is qualified, reduce testing cost.
Area normalization method calculates total crocin and two methods of-II content of calculating crocin-I and glycosides are commented in a survey more
In conjunction with realizing the assay that a reference substance completes each crocin, method is easy, as a result reliably, each western red in sample
The difference of proportional quantities between flower glycosides component can reflect west safflower growth and development, pick and process, the processes such as storage to a certain extent
The transformation characteristics of middle crocin constituents provide more to improve west safflower quality in west safflower production and processing and the process of circulation
Good Technical Reference.
Several preferred embodiments of the invention have shown and described in above description, but as previously described, it should be understood that the present invention
It is not limited to form disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other
Combination, modification and environment, and in this application in the invented the scope of the idea, above-mentioned introduction or the skill of related fields can be passed through
Art or knowledge are modified.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present invention, then all
It should be within the scope of protection of the appended claims of the present invention.
Claims (4)
1. a kind of method of crocin constituents in area normalization method measurement west safflower medicinal material, which is characterized in that including step
It is rapid:
A. determine chromatographic condition, chromatographic condition be UltimateXBC18 chromatographic column, chromatographic column be 250mm × 4.6mm, 5 μm；Column
Temperature: 30 DEG C；Mobile phase: water-methanol, gradient elution, 0-60min, methanol: 20%-100%；DAD detector wavelength range 200-
600nm, Detection wavelength 440nm, flow velocity: 1.0mLmin-1；Sample volume: 10 μ L；
B. reference substance solution is prepared:
Mixed reference substance solution: precision weighs-II reference substance of-I reference substance of crocin and crocin, is placed in brown measuring bottle
In, scale is dissolved and be settled to Diluted Alcohol, obtains mixed reference substance solution, is shaken up, it is spare；
- I reference substance solution of crocin: precision weighs-I reference substance of crocin and sets in brown measuring bottle, simultaneously with Diluted Alcohol dissolution
It is settled to scale, obtains-I reference substance solution of crocin；
C. it prepares test solution: referring under Chinese Pharmacopoeia version west safflower in 2015, weighing west safflower sample powder about
10mg, accurately weighed, set in 50mL brown measuring bottle, add Diluted Alcohol 40mL, ice-bath ultrasonic: 250W, 40KHz handle 20min, place
Room temperature adds ethyl alcohol to be settled to scale, shakes up, and 0.22 μm of miillpore filter filtration takes subsequent filtrate, obtains test solution；
D. according to the content of the quantified by external standard method crocin-I of version Chinese Pharmacopoeia in 2015 and glycosides-II；
E. one the contents for commenting method QMSA measurement crocin-I and glycosides-II are surveyed more；
F. the content of the total crocin of determined by ultraviolet spectrophotometry；
G. area normalization method measures the content of total crocin, and precision draws reference substance solution in test solution and step b
Each 10 μ L injects hplc determination, and minute is no less than 60 minutes, is analyzed by mass spectrometry simultaneously, Mass Spectrometry Conditions are as follows:
Ion source: atmospheric pressure electrospray ion source；Detection pattern: negative ions；Full scan karyoplasmic ratio: 50-1200 m/z；Dry temperature
Degree: 350 DEG C, dry gas stream amount: 12.0Lmin-1, atomization gas pressure: 35.0psi；Capillary voltage: 4.5kV；Collision voltage
1.0V；0.4mLmin-1 is branched into mass spectrographic flow rate of mobile phase, is control with crocin-I, by the total glycosides of C=(C glycosides-
The total glycosides of I × A)/A glycosides-I calculates the content of total crocin,
Wherein A glycosides-I is-I chromatographic peak peak area of crocin in sample；The total glycosides of A is west safflower sample 440nm lower 12 western red
The sum of flower glycoside chromatographic peak peak area；C glycosides-I is the mass concentration of crocin-I in the west safflower sample of external standard method；C
Total glycosides is total crocin mass concentration in west safflower sample.
2. the method for crocin constituents, special in area normalization method measurement west safflower medicinal material as described in claim 1
Sign is, the step d, further for, take crocin-I and glycosides-II in step b mixed reference substance solution 1,2,4,8,
10,20 μ L, by chromatographic condition in step a, sample introduction is analyzed, peak area is measured, respectively with-II mass concentration of crocin-I and glycosides
Linear regression is carried out to its corresponding peak area, obtains regression equation, precision measures the 10 μ L of test solution in step c, injects liquid
Chromatography measurement, corresponding peak area substitute into regression equation calculation glycosides-I and-II content of glycosides.
3. the method for crocin constituents, special in area normalization method measurement west safflower medicinal material as described in claim 1
Sign is, the step e, further for, take mixed reference substance solution in step b, respectively 1,2,4,8,10,15,20 μ L of sample introduction,
Peak area is recorded, using Supplements method, with crocin-I for internal reference object, calculates-I pair of crocin-II of crocin
Relative correction factor fk/s, fk/s calculation formula: fk/s=(Cs × Ak)/(Ck × As) ,-II quality of ingredient crocin to be measured
Concentration calculation formula: Ck '=(Cs × Ak ')/(fk/s × As),
Wherein Cs is-I mass concentration of internal reference object crocin, and As is that internal reference looks for spectral peak peak area, and Ck is-II pair of crocin
According to quality concentration, Ak is-II reference substance chromatographic peak peak area of crocin, and Ck ' is-II matter of crocin in west safflower sample
Concentration is measured, Ak ' is-II chromatographic peak peak area of crocin in west safflower sample.
4. the method for crocin constituents, special in area normalization method measurement west safflower medicinal material as described in claim 1
Sign is, the step f, further draws-I reference substance solution 0.1 of crocin for, precision, 0.2,0.4,0.6,0.8,
1.0mL is placed in 10mL measuring bottle, is diluted to scale with Diluted Alcohol, makees blank control with Diluted Alcohol, using UV-vis spectroscopy light
Degree method measures absorbance at 440nm wavelength, obtains standard curve；Test solution 5mL in accurate aspiration step c, sets 50mL
In brown measuring bottle, it is diluted to scale with Diluted Alcohol, measures absorbance, Yi Xihong at 440nm wavelength with visible spectrophotometry
Flower glycosides-I is reference standards, calculates total crocin content by standard curve.
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| CN112082960A (en) * | 2019-06-13 | 2020-12-15 | 上海崇明农联西红花研究发展中心 | Detection method for content of crocin |
| CN115389639A (en) * | 2021-05-24 | 2022-11-25 | 赣江中药创新中心 | Crocin compound targeted identification method and novel crocin compound |
| CN113533221B (en) * | 2021-07-27 | 2024-05-24 | 复旦大学 | Online testing equipment and method for detecting content of effective components of crocus sativus filaments |
| CN114324701B (en) * | 2021-12-21 | 2023-06-30 | 中南大学 | Method for rapidly and simultaneously determining content of crocin-1, crocin-2, crocin-3 and crocin-4 |
| CN115407004A (en) * | 2022-08-31 | 2022-11-29 | 舟山市食品药品检验检测研究院 | Saffron quality evaluation method based on one-test-multiple evaluation |
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