CN104897809B - The assay method of Chinese medicine Fructus Rubi middle finger index composition content - Google Patents
The assay method of Chinese medicine Fructus Rubi middle finger index composition content Download PDFInfo
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Abstract
The embodiment of the invention discloses the assay method of a kind of Chinese medicine Fructus Rubi middle finger index composition content, comprise the following steps: (1) prepares need testing solution；(2) preparation mixing reference substance solution；(3) Ultra Performance Liquid Chromatography detection；(4) the N respective standard curve of kind index components is set up；(5) N kind index components respective content in test sample is determined.The assay method of the Chinese medicine Fructus Rubi middle finger index composition content that the present invention builds, precise and high efficiency, can be used for the quality control to Chinese medicine Fructus Rubi, and the method can detect many indexes composition in Chinese medicine Fructus Rubi simultaneously, the method detected respectively relative to different index components, shorten detection total time, save reagent cost.And testing result is accurate, precision is high, reproducible, and stability is high, and specificity is strong.
Description
Technical field
The present invention relates to Chinese medicine quality control field, particularly to the mensuration side of Chinese medicine Fructus Rubi middle finger index composition content
Method.
Background technology
Chinese medicine Fructus Rubi is the dry fruit of Rosaceae rubus rubus chingii Hu (Rubus chingii Hu).
Early summer fruit by green change greenish-yellow time gather, remove stalk, leaf, put in boiling water and slightly to scald or slightly to steam, take out, be dried, obtain Chinese medicine and cover basin
Son.Chinese medicine Fructus Rubi has effect of supplementing the kidney to control the nocturnal reducing urination, nourishing the liver to improve visual acuity, is clinically used for treating emission and spermatorrhea, enuresis frequent micturition, sun
Flaccidity premature ejaculation, mesh are the most dim-sighted.Modern pharmacological research shows that Fructus Rubi has antioxidation, antiinflammatory, antithrombotic, anticancer isoreactivity, its
Chemical composition mainly has flavonoid, terpenoid, steroid, phenolic acids etc..
" Chinese Pharmacopoeia " version one in 2010 has included Chinese medicine Fructus Rubi character and powder differentiates, do not include thin layer differentiate and
Assay item.But, only it is difficult to scientific evaluation by the discriminating of character and powder and controls the quality of Chinese medicine Fructus Rubi.
The most further measure the content of index components in Chinese medicine Fructus Rubi, could fundamentally ensure the drug effect of Chinese medicine Fructus Rubi.
Summary of the invention
In order to preferably evaluate and control the quality of Chinese medicine Fructus Rubi, the embodiment of the invention discloses Chinese medicine Fructus Rubi middle finger
The assay method of index composition content.Technical scheme is as follows:
The assay method of Chinese medicine Fructus Rubi middle finger index composition content, comprises the following steps:
(1) need testing solution is prepared
By Chinese medicine Fructus Rubi that quality is M and V volume 0～100% methanol mixed, obtain the two mixed liquor, mixed determining
Close weight M of liquid1After, to its carry out extract operation, then with 0～100% methanol the weight of described mixed liquor is complemented to M1,
Take supernatant, obtain need testing solution；
(2) preparation mixing reference substance solution
Weigh the reference substance of N kind index components in Chinese medicine Fructus Rubi respectively, respectively with 0～100% methanol dissolve, constant volume,
Obtain the reference substance solution with each index components of concentration known；
Measure the reference substance solution of each index components the most respectively, mixing, with 0～100% methanol constant volume, obtain mixing and compare
Product storing solution, in described mixing reference substance storing solution, each index components has concentration known；
By reference substance mixing storing solution with 0～100% methanol stepwise dilution become a series of and be respectively provided with different concentration known
Mixing reference substance solution；
(3) Ultra Performance Liquid Chromatography detection
Chromatographic condition includes: fixing phase: use the chromatographic column of octadecylsilane chemically bonded silica；Flowing phase: A phase is second
Nitrile, B phase is 0.1% formic acid, A phase: B phase is (3～70): (97～30)；Flow velocity is 0.2～1.0mL/min, and column temperature is 30～50
DEG C, PDA detector, detection wavelength is 253～344nm；
Under described chromatographic condition, by V1The need testing solution of volume and the mixing reference substance solution of each concentration are injected separately into
In Ultra Performance Liquid Chromatography instrument, it is thus achieved that the Ultra Performance Liquid Chromatography figure of need testing solution and each concentration mix the super of reference substance solution
High-efficient liquid phase chromatogram；
(4) the N respective standard curve of kind index components is set up
In the Ultra Performance Liquid Chromatography figure of each mixing reference substance solution, the chromatographic peak peak area of each index components is sat for vertical
Mark or abscissa, to mix in reference substance solution the concentration of each index components as abscissa or vertical coordinate, set up each index respectively
The standard curve of composition；
(5) N kind index components respective content in test sample is determined
According to N kind index components respective chromatographic peak peak area in need testing solution Ultra Performance Liquid Chromatography figure and built
The standard curve of vertical each index components, calculates the N respective concentration C of kind index components respectivelyDense, and distinguish according to the following formula
Calculate the respective content of N kind index components in test sample:
Content=CDense*V/M。
In the preferred embodiment of the present invention, supersound extraction it is extracted as described in.
In the preferred embodiment of the present invention, the weight of described mixed liquor is being complemented to M1After, centrifugal, then take
Supernatant.
In the preferred embodiment of the present invention, described 0～100% methanol be 70～85% methanol.
In the preferred embodiment of the present invention, described flow velocity is 0.4mL/min.
In the preferred embodiment of the present invention, described flowing phase: A phase is acetonitrile, B phase is 0.1% formic acid, A phase:
B phase is 18:82.
In the one more preferably embodiment of the present invention, described index components is ellagic acid, i.e. during N=1, and described color
Spectral condition includes: fixing phase: use the chromatographic column of octadecylsilane chemically bonded silica；Flowing phase: A phase is acetonitrile, and B phase is
0.1% formic acid, A phase: B phase is 18:82；Flow velocity 0.4mL/min；Column temperature 40 DEG C；PDA detector；Detection wavelength is 253nm.
In the one more preferably embodiment of the present invention, described index components is ellagic acid and Kaempferol-O-Folium Symplocoris Caudatae
Glucosides, i.e. during N=2, described chromatographic condition includes: fixing phase: use the chromatographic column of octadecylsilane chemically bonded silica；Flowing
Phase: A phase is acetonitrile, B phase is 0.1% formic acid, A phase: B phase is 18:82；Flow velocity 0.4mL/min；Column temperature 40 DEG C；PDA detector；
Detection wavelength is 253nm and 265nm.
In the one more preferably embodiment of the present invention, described index components is adenosine, gallic acid, brevifolin
Acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside, tiliroside, i.e. N=8
Time, described chromatographic condition includes: fixing phase: use the chromatographic column of octadecylsilane chemically bonded silica；Flowing phase: acetonitrile: 0.1%
Formic acid ratio is 0～2min, 3:97～5:95；2～8min, 5:95～11.5:88.5；8～13.5min, 11.5:88.5～
15.5:84.5；13.5～15min, 15.5:84.5～18:82；15～18min, 18:82～20:80；18～23min, 20:80
～38:62；23～25min, 38:62～70:30；Column temperature is 30 DEG C；PDA detector；Detection wavelength is 256nm, 265nm,
271nm and 344nm.
The assay method of the Chinese medicine Fructus Rubi middle finger index composition content that the present invention builds, precise and high efficiency, can be used for Chinese medicine
The quality control of Fructus Rubi, and the method can detect many indexes composition in Chinese medicine Fructus Rubi, relative to difference simultaneously
The method that index components detects respectively, shortens detection total time, saves reagent cost.And testing result is accurate, precision is high,
Reproducible, stability is high, and specificity is strong.
Certainly, arbitrary product or the method for implementing the present invention it is not absolutely required to reach all the above excellent simultaneously
Point.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
In having technology to describe, the required accompanying drawing used is briefly described, it should be apparent that, the accompanying drawing in describing below is only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, it is also possible to
Other accompanying drawing is obtained according to these accompanying drawings.
Figure 1A is ellagic acid reference substance solution Ultra Performance Liquid Chromatography figure under 253nm wavelength；
Figure 1B is the chromatogram of Chinese medicine Fructus Rubi (Zhejiang) need testing solution under 253nm wavelength；
Fig. 2 A is ellagic acid and Kaempferol-O-rutinoside mixing reference substance solution chromatogram under 253nm wavelength；
Fig. 2 B is ellagic acid and Kaempferol-O-rutinoside mixing reference substance solution chromatogram under 265nm wavelength；
Fig. 2 C is Chinese medicine Fructus Rubi (Zhejiang) need testing solution chromatogram under 253nm wavelength；
Fig. 2 D is Chinese medicine Fructus Rubi (Zhejiang) need testing solution chromatogram under 265nm wavelength；
Fig. 3 A is the lower 8 kinds of mixing reference substance solution chromatograms of 256nm wavelength；
Fig. 3 B is the lower 8 kinds of mixing reference substance solution chromatograms of 265nm wavelength；
Fig. 3 C is the lower 8 kinds of mixing reference substance solution chromatograms of 271nm wavelength；
Fig. 3 D is the lower 8 kinds of mixing reference substance solution chromatograms of 344nm wavelength；
Fig. 3 E is the chromatogram of Chinese medicine Fructus Rubi (Zhejiang) need testing solution under 256nm wavelength；
Fig. 3 F is the chromatogram of Chinese medicine Fructus Rubi (Zhejiang) need testing solution under 265nm wavelength；
Fig. 3 G is the chromatogram of Chinese medicine Fructus Rubi (Zhejiang) need testing solution under 271nm wavelength；
Fig. 3 H is the chromatogram of Chinese medicine Fructus Rubi (Zhejiang) need testing solution under 344nm wavelength.
Detailed description of the invention
" Chinese Pharmacopoeia " version one in 2010 has included Chinese medicine Fructus Rubi character and powder differentiates, do not include thin layer differentiate and
Assay item.It is currently known adenosine, gallic acid, brevifolin carboxylic acid, progallin A, tan in Chinese medicine Fructus Rubi composition
The content of flower acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside is to evaluation Chinese medicine Fructus Rubi medicine
Property is critically important.
The present invention establishes the assay method of a kind of Chinese medicine Fructus Rubi middle finger index composition content.The method can be according to ability
The needs of field technique personnel, using one or more in Chinese medicine Fructus Rubi composition as index components, carry out assay, at this
The kind of index components is not specifically limited by inventor.
In the inventive solutions, prepare Chinese medicine Fructus Rubi and 0～100% methanol in the step of need testing solution
After mixing, weigh, after extraction, be re-weighed, supply weightlessness with the methanol of same volume mark, then take supernatant, above-mentioned steps
Can reach two purposes: fully extract and constant volume.This constant volume method relative to exhausting supernatant again by the method for Extraction solvent constant volume,
Can eliminate and supernatant artificially all can not be exhausted produced error during experimental implementation, it is thus achieved that result closer to reason
Opinion actual value.This constant volume method is the improvement to traditional constant volume method.After the method constant volume, take part supernatant, it is possible to
Meet the demand of subsequent experimental, simplify the experimental procedure of constant volume.Those skilled in the art are it is conceivable that 0～100% first
Alcohol can realize the purpose index components in Chinese medicine Fructus Rubi extracted, but in order to improve extraction efficiency, preferably 70～
85% methanol is as Extraction solvent, and more preferably volume fraction is that 70% methanol extracts；In order to ensure when taking supernatant, will not
Get medicinal residues, affect follow-up testing result, after being preferably centrifuged, take supernatant.
Technical scheme, during preparation reference substance solution, it is fixed first to be dissolved respectively by index components reference substance
Hold, make the reference substance storing solution with concentration known；Respectively precision draws appropriate reference substance storing solution again, mixing, with 0～
100% methanol constant volume, obtains the mixing reference substance solution with each index components of concentration known；Finally, stepwise dilution becomes one
Series is respectively provided with the mixing reference substance solution of different concentration known.So can realize detecting several reference substance solution simultaneously
Purpose, saves the tedious steps for each reference substance sample introduction respectively, time-consuming.In the art, generally dilution 5-8 is dense
Degree gradient, so processes when Criterion curve, and linear relationship is good, can correctly evaluate index components in need testing solution
Concentration.
The chromatographic condition of technical solution of the present invention: fixing phase: the specification of octadecylsilane chemically bonded silica (C18) chromatographic column
For 3.0mm × 100mm, 1.7 μm；Flowing phase: A phase is acetonitrile, and B phase is 0.1% formic acid, when analyzing one or two kind of index components,
Can set isocratic elution, i.e. A phase is constant with B Phase Proportion, when analyzing more kinds of index components, in order to various index components be filled
Separate, it is ensured that the composition of each chromatographic peak is single, may be set to gradient elution, i.e. A phase and B Phase Proportion are along with when setting eluting
Between and change.In the preferred version of the present invention, when the index components of Chinese medicine Fructus Rubi of detection be ellagic acid or ellagic acid and
During Kaempferol-O-rutinoside, flow phase: acetonitrile: 0.1% formic acid is 18:82；When detection 8 kinds of indexs of Chinese medicine Fructus Rubi become
Point (adenosine, gallic acid, brevifolin carboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, nimbecetin-
3-O-glucoside, tiliroside, described reference substance is adenosine, gallic acid, brevifolin carboxylic acid, progallin A, tan
Flower acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside) time, flow phase: acetonitrile: 0.1% formic acid
Ratio is 0～2min, 3:97～5:95；2～8min, 5:95～11.5:88.5；8～13.5min, 11.5:88.5～15.5:
84.5；13.5～15min, 15.5:84.5～18:82；15～18min, 18:82～20:80；18～23min, 20:80～38:
62；23～25min, 38:62～70:30.Flow velocity can be 0.2～1.0mL/min, preferably 0.4mL/min, column temperature 30～50
℃.The present invention is directed to the chromatographic test strip part that index components in Chinese medicine Fructus Rubi sets, it is thus achieved that chromatogram display index components
Chromatographic peak peak shape sharp-pointed, do not trail, the composition in test sample can be completely separated, detect data true and accurate.The present invention selects
PDA detector, i.e. photodiode array detector, can at multiple wavelengths material be detected, detection knot simultaneously
The most reliable and the most stable.
Heretofore described 0～100% methanol refer to the methanol aqueous solution that volume fraction is 0～100%, such as 70% first
Alcohol refers to the methanol aqueous solution that volume fraction is 70%, and 85% methanol refers to the methanol aqueous solution that volume fraction is 85%；Equally
Ground, described 0.1% formic acid be volume fraction be the aqueous formic acid of 0.1.
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Describe, it is clear that described embodiment is only a part of embodiment of the present invention rather than whole embodiments wholely.Based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under not making creative work premise
Embodiment, broadly falls into the scope of protection of the invention.
Instrument and reagent
Ultra Performance Liquid Chromatography instrument (Waters ACQUITY UPLCTMSystem, including column oven, online vacuum outgas
Machine, high pressure binary gradient pump, automatic sampler, PDA detector, Waters, US)；100000/balance (METTLER
TOLEDO XP6, METTLER company of Switzerland)；Ten thousand/balance (METTLER TOLEDO AL204；Switzerland METTLER is public
Department)；Ultrasonic cleaner (HS6150D, Tianjin perseverance AudioCodes skill Development Co., Ltd)；Water purification machine (Millipore company of the U.S.).
Methanol is chromatographically pure (Tianjin Concord Technology Co., Ltd.)；Acetonitrile is chromatographically pure (U.S. Sigma-Aldrich
Company)；Water is self-control ultra-pure water.
Adenosine, the reference substance of gallic acid are purchased from National Institute for Food and Drugs Control；Brevifolin carboxylic acid, gallic acid
Ethyl ester, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside, the reference substance of tiliroside are self-control.Self-control
Reference substance preparation process: Chinese medicine Fructus Rubi 95% ethanol and heating and refluxing extraction 2 times respectively of 60% ethanol, each 2h, merges
Extracting solution, recovered under reduced pressure obtains total extractum.Add appropriate distilled water by total extractum suspendible, successively by petroleum ether, dichloromethane, second
Acetoacetic ester and n-butanol extraction.Acetic acid ethyl ester extract separates through silica gel column chromatography repeatedly, gel column chromatography, preparative liquid chromatography
Obtain tiliroside, ellagic acid and progallin A.N-butyl alcohol extract through D101 macroporous resin column separate, respectively with water,
30%, the ethanol of 50%, 70%, 95% carries out gradient elution, and n-butyl alcohol 30% ethanol elution part separates through silica gel column chromatography
Obtain brevifolin carboxylic acid；N-butyl alcohol 50% ethanol elution part is through silica gel column chromatography, preparative liquid chromatography isolated Rhizoma Kaempferiae
Phenol-3-O-rutinoside and Kampferol-3-O-glucoside.The reference substance obtained made above is pure through high-efficient liquid phase color spectral test
Degree, and confirm its structure by NMR (Nuclear Magnetic Resonance) spectrum and Mass Spectrometer Method.
Chinese medicine Fructus Rubi (Zhejiang, the place of production), Chinese medicine Fructus Rubi (Shandong, the place of production), Chinese medicine Fructus Rubi (Henan, the place of production), Chinese medicine cover
Basin (Anhui, the place of production), Chinese medicine Fructus Rubi (Beijing, the place of production), Chinese medicine Fructus Rubi (Hubei, the place of production), Chinese medicine Fructus Rubi (river, the place of production
North).
Term " accurately weighed " mentioned in the present invention: accurately weighed mean weigh weight should be accurately to taken weight
One thousandth." accurate draw ": mean that measuring the accuracy of volume should meet in national standard the degree of accuracy to this measurement instrument
Requirement.
In the art, the relative standard deviation (RSD) of the experimental data of assay, within 5%, illustrates experiment knot
Fruit is reliable, can react the content of something truly.
In the art, average recovery, in the range of 95～105%, illustrates that abnormal phenomena does not occurs in experiment link, institute
The method set up meets assay requirement, and the experimental result obtained is reliable.
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1
(1) preparation of need testing solution
Chinese medicine Fructus Rubi to 7 places of production is cooked following process respectively:
Chinese medicine Fructus Rubi is pulverized, crosses 40 mesh sieves, accurately weighed 0.5g, put in conical flask, accurate addition 70% methanol
50mL, close plug, weigh 44.1g, room temperature supersound process 30min, is re-weighed, and supplies the weight of loss with 70% methanol aqueous solution,
Shake up, centrifugal 14000 turns of 10min, take supernatant 1mL.
Each process is repeated 3 times.
(2) preparation of reference substance solution
Accurately weighed ellagic acid reference substance 7.75mg, adds 100% methanol and dissolves and be settled to 25mL, be configured to 0.31mg
mL-1Reference substance storing solution.
Accurate absorption ellagic acid reference substance storing solution 1.5mL, and by 100% methanol constant volume to 5mL, it is configured to 93.00 μ
g·mL-1Reference substance solution, be that 100% methanol solution gradually dilutes by 1mL volume fraction, the concentration of dilution is respectively 46.50
μg·mL-1、23.25μg·mL-1、11.63μg·mL-1、5.81μg·mL-1、2.91μg·mL-1、1.45μg·mL-1。
(3) Ultra Performance Liquid Chromatography detection
Chromatographic condition: solid phase: ACQUITY BEH C18 (3.0mm × 100mm, 1.7 μm) chromatographic column；Flowing phase: A phase: second
Nitrile and B phase: the ratio of 0.1% formic acid is 18:82；Flow velocity: 0.4mL min-1；Column temperature 40 DEG C；PDA detector, detection wavelength is
253nm。
Precision draws need testing solution and the ellagic acid reference substance solution 3 μ L of variable concentrations respectively, injects ultra high efficiency liquid phase
Chromatograph (UPLC) detects.The chromatogram of ellagic acid reference substance solution is as shown in Figure 1A.The chromatogram of need testing solution
As a example by the chromatogram (Figure 1B) of the Chinese medicine Fructus Rubi need testing solution in the place of production, Zhejiang.
Can be seen that under 253nm detection wavelength from Figure 1A, the chromatographic peak that chromatographic peak is ellagic acid that 2.70min occurs,
The i.e. retention time of ellagic acid is 2.70min.Correspondingly, in fig. ib, the chromatographic peak that 2.70min occurs is i.e. the place of production, Zhejiang
The chromatographic peak of ellagic acid in Chinese medicine Fructus Rubi need testing solution, and realize good separation with other compositions.
(4) standard curve of ellagic acid is set up
In reference substance solution, the concentration of ellagic acid is as abscissa, with tan in the Ultra Performance Liquid Chromatography figure of reference substance solution
The chromatographic peak peak area of flower acid is vertical coordinate, sets up the standard curve of ellagic acid, with signal to noise ratio (S/N) 10 as quantitative limit, and result
It is shown in Table 1.As can be known from Table 1, ellagic acid is at 1.45～93 μ g mL-1In concentration range, standard curve linear relationship is good.
The equation of linear regression of table 1 ellagic acid, correlation coefficient, the range of linearity, quantitative limit
(5) ellagic acid content in test sample is determined
According to the chromatographic peak peak area of ellagic acid in need testing solution Ultra Performance Liquid Chromatography figure, and the ellagic acid set up
Standard curve, calculate the concentration of ellagic acid, and calculate the content of ellagic acid in test sample according to the following formula:
Content=CDense*V/M。
Obtain the content of index components ellagic acid in the Chinese medicine Fructus Rubi in 7 places of production according to this formula, the results are shown in Table 2.
Index components ellagic acid assay result (Mean ± SD, n=3) in table 2 Chinese medicine Fructus Rubi
Precision, stability, repeatability and average recovery is carried out below as a example by the Chinese medicine Fructus Rubi as Zhejiang of the place of production
Test.
(6) precision test
Need testing solution is prepared, by embodiment 1 step (3) chromatographic condition, continuous sample introduction 6 by the step (1) of embodiment 1
Secondary, each 3 μ L, in detection need testing solution, the content of ellagic acid, evaluates the withinday precision of ellagic acid, the results are shown in Table 3.Press
Embodiment 1 step (1) prepares need testing solution, by embodiment 1 step (3) chromatographic condition, and continuous three days sample introductions, 6 times/day, 3 μ
L/ time, measure, evaluate the day to day precision of ellagic acid, the results are shown in Table 4.Understand from table 3 and table 4, in a few days and in the daytime accurate
The RSD of the ellagic acid content that degree test obtains is respectively 0.33%, 2.31%.Result shows that ellagic acid precision is good, meets
The requirement of assay.
The withinday precision result of the test (mg/g, n=6) of table 3 ellagic acid
The day to day precision result of the test (mg/g, n=3) of table 4 ellagic acid
(7) stability test
The accurately weighed Chinese medicine Fructus Rubi 0.5g pulverizing 40 mesh, prepares test sample by the method for embodiment 1 step (1) molten
Liquid, by the chromatographic condition of embodiment 1 step (3), respectively at 0,2,4,8,10 and 12h sample introduction 3 μ L, measures, investigates test sample molten
In liquid, the stability of ellagic acid 12h, the results are shown in Table 5.As known from Table 5, the relative standard deviation (RSD) of ellagic acid content is
2.24%.Result is basicly stable in showing the 12h at ambient temperature of the ellagic acid in need testing solution.
Table 5 ellagic acid stability test result (mg/g, n=6)
(8) replica test
The accurately weighed Chinese medicine Fructus Rubi 0.5g pulverizing 40 mesh, by the method parallel processing 6 parts of embodiment 1 step (1)
Need testing solution, by the chromatographic condition of embodiment 1 step (3) sample introduction 3 μ L respectively, measures, the results are shown in Table 6.As known from Table 6, tan
The RSD of flower acid content is 0.49%.Result shows that the detection method repeatability of the present invention is good.
Table 6 ellagic acid replica test result (mg/g, n=6)
(9) average recovery test
The accurately weighed Chinese medicine Fructus Rubi 0.25g pulverizing 40 mesh, the most accurately adds and ellagic acid amount phase in test sample
When ellagic acid reference substance storing solution (ellagic acid reference substance Stock concentrations 0.31mg mL-1, mark-on volume 1mL), by implementing
6 parts of test samples of the method parallel processing of example 1 step (1), by the chromatographic condition of embodiment 1 step (3) sample introduction 3 μ L respectively, measure,
Calculate average recovery, the results are shown in Table 7.
The average recovery result of the test (n=6) of index components ellagic acid in table 7 Chinese medicine Fructus Rubi
Embodiment 2
(1) preparation of need testing solution
With embodiment 1 step (1).
(2) preparation of reference substance solution
Accurately weighed ellagic acid reference substance and Kaempferol-O-rutinoside reference substance 7.75mg, 8.18mg respectively, respectively
Add 100% methanol dissolve and be settled to 25mL, 10mL respectively, be configured to 0.31mg mL respectively-1Ellagic acid reference substance storing solution
With 0.82mg mL-1Kaempferol-O-rutinoside reference substance storing solution.
Respectively precision draw ellagic acid reference substance storing solution and Kaempferol-O-rutinoside reference substance storing solution 1.5mL,
0.2mL mixes, and by 100% methanol constant volume to 5mL, is configured to 93.00 μ g mL-1Ellagic acid, 32.72 μ g mL-1Rhizoma Kaempferiae
Phenol-3-O-rutinoside mixing reference substance solution, is 100% methanol solution stepwise dilution by 1mL volume fraction respectively, ellagic acid
Concentration is respectively 46.50,23.25,11.63,5.81,2.91,1.45 μ g mL-1, Kaempferol-O-rutinoside concentration is respectively
It is 16.36,8.18,4.09,2.05,1.02,0.51 μ g mL-1。
(3) Ultra Performance Liquid Chromatography detection
Chromatographic condition: fixing phase: ACQUITY BEH C18 (3.0mm × 100mm, 1.7 μm) chromatographic column；Flowing phase: A phase:
Acetonitrile and B phase: the ratio of 0.1% formic acid is 18:82；Flow velocity: 0.4mL min-1；Column temperature 40 DEG C；PDA detector, detects wavelength
For 253nm and 265nm.
Precision draws need testing solution and the ellagic acid of variable concentrations and the mixing comparison of Kaempferol-O-rutinoside respectively
Product solution 3 μ L, injects in Ultra Performance Liquid Chromatography instrument (UPLC) and detects.Ellagic acid and Kaempferol-O-rutinoside pair
Chromatogram according to product solution is distinguished the most as shown in Figure 2 A and 2 B.The chromatogram of need testing solution is with the Chinese medicine Fructus Rubi in the place of production, Zhejiang
As a example by the chromatogram (Fig. 2 C and Fig. 2 D) of need testing solution.
From Fig. 2 A it can be seen that under 253nm wavelength, the retention time of ellagic acid is 2.70min.Correspondingly, Fig. 2 C
In, the chromatographic peak at 2.70min be exactly the place of production, Zhejiang Chinese medicine Fructus Rubi need testing solution in the chromatographic peak of ellagic acid, and with
Other compositions realize good separation.From Fig. 2 B it can be seen that under 265nm wavelength, the reservation of Kaempferol-O-rutinoside
Time is 3.85min.Correspondingly, in Fig. 2 D, the chromatographic peak at 3.85min is exactly the Chinese medicine Fructus Rubi test sample in the place of production, Zhejiang
The chromatographic peak of Kaempferol-O-rutinoside in solution, and realize good separation with other compositions.
(4) ellagic acid, the standard curve of Kaempferol-O-6-O-.alpha.-L-rhamnosyl-D-glucose. are set up respectively
In mixing reference substance solution, the concentration of ellagic acid reference substance is as abscissa, to mix the ultra high efficiency of reference substance solution
In liquid chromatogram, the chromatographic peak peak area of ellagic acid is vertical coordinate, sets up the standard curve of ellagic acid.
In mixing reference substance solution, the concentration of Kaempferol-O-rutinoside reference substance is as abscissa, to mix comparison
In the Ultra Performance Liquid Chromatography figure of product solution, the chromatographic peak peak area of Kaempferol-O-rutinoside is vertical coordinate, sets up Rhizoma Kaempferiae
The standard curve of phenol-3-O-rutinoside.
The two the results are shown in Table 8 all with signal to noise ratio (S/N) 10 as quantitative limit.As can be known from Table 8, two indices composition is respectively
From concentration range internal linear relation good.
Table 8 ellagic acid and the equation of linear regression of Kaempferol-O-rutinoside, correlation coefficient, the range of linearity, quantitative limit
(5) ellagic acid and Kaempferol-O-rutinoside respective content in test sample are determined
Respectively according to ellagic acid in need testing solution Ultra Performance Liquid Chromatography figure and the chromatograph of Kaempferol-O-rutinoside
Peak-to-peak area, and the ellagic acid set up and the standard curve of Kaempferol-O-rutinoside, calculate ellagic acid and Rhizoma Kaempferiae respectively
The concentration of phenol-3-O-rutinoside, and calculate ellagic acid and Kaempferol-O-rutinoside in test sample according to the following formula
Content:
Content=CDense*V/M。
Index components ellagic acid and Kaempferol-O-6-O-.alpha.-L-rhamnosyl-D-glucose. in the Chinese medicine Fructus Rubi in 7 places of production is obtained according to this formula
The content of glycosides, the results are shown in Table 9.
Index components ellagic acid in table 9 Chinese medicine Fructus Rubi, Kaempferol-O-rutinoside assay result (Mean ±
SD, n=3)
Precision, stability, repeatability and average recovery is carried out below as a example by the Chinese medicine Fructus Rubi as Zhejiang of the place of production
Test.
(6) precision test
The accurately weighed Chinese medicine Fructus Rubi 0.5g pulverizing 40 mesh, prepares test sample by the method for embodiment 1 step (1) molten
Liquid, by embodiment 2 step (3) chromatographic condition, continuous sample introduction 6 times, each 3 μ L, ellagic acid, Rhizoma Kaempferiae in detection need testing solution
The content of phenol-3-O-rutinoside, evaluates ellagic acid, the withinday precision of Kaempferol-O-rutinoside, the results are shown in Table
10.Need testing solution is prepared by embodiment 1 step (1), by embodiment 1 step (3) chromatographic condition, continuous three days sample introductions, 6 times/
My god, 3 μ L/ time, measure, evaluate ellagic acid, the day to day precision of Kaempferol-O-rutinoside, the results are shown in Table 11.From table 10
Understanding with 11, ellagic acid, the RSD of Kaempferol-O-rutinoside content that withinday precision test obtains are respectively 0.33%
With 1.08%, ellagic acid, the RSD of Kaempferol-O-rutinoside content that day to day precision test obtains are respectively 2.31%
With 0.89%.Result shows that ellagic acid, Kaempferol-O-rutinoside precision are good, meets the requirement of assay.
The withinday precision result of the test (mg/g, n=6) of table 10 ellagic acid and Kaempferol-O-rutinoside
The day to day precision result of the test (mg/g, n=3) of table 11 ellagic acid and Kaempferol-O-rutinoside
(7) stability test
The accurately weighed Chinese medicine Fructus Rubi 0.5g pulverizing 40 mesh, prepares test sample by the method for embodiment 1 step (1) molten
Liquid, by the chromatographic condition of embodiment 2 step (3), respectively at 0,2,4,8,10 and 12h sample introduction 3 μ L, measures, investigates test sample molten
In liquid, ellagic acid, the stability of Kaempferol-O-rutinoside 12h, the results are shown in Table 12.As known from Table 12, ellagic acid, Rhizoma Kaempferiae
The RSD of phenol-3-O-rutinoside content is respectively 2.24%, 1.90%.Result shows the ellagic acid in need testing solution and mountain
In naphthol-3-O-rutinoside 12h at ambient temperature basicly stable.
Table 12 ellagic acid and Kaempferol-O-rutinoside stability test result (mg/g, n=6)
(8) replica test
The accurately weighed Chinese medicine Fructus Rubi 0.5g pulverizing 40 mesh, by the method parallel processing 6 parts of embodiment 1 step (1)
Need testing solution, by the chromatographic condition of embodiment 2 step (3) sample introduction 3 μ L respectively, measures, the results are shown in Table 13.As known from Table 13,
Ellagic acid, the RSD of Kaempferol-O-rutinoside content are respectively 0.49%, 0.42%.Result shows the detection side of the present invention
Method repeatability is good.
Table 13 ellagic acid and Kaempferol-O-rutinoside replica test result (mg/g, n=6)
(9) average recovery test
The accurately weighed Chinese medicine Fructus Rubi 0.25g pulverizing 40 mesh, the most accurately adds and ellagic acid, Rhizoma Kaempferiae in test sample
(ellagic acid reference substance stores up for ellagic acid that phenol-3-O-rutinoside amount is suitable, Kaempferol-O-rutinoside reference substance storing solution
Standby liquid concentration 0.31mg mL-1, mark-on volume 1mL；Kaempferol-O-rutinoside reference substance Stock concentrations 0.82mg
mL-1, mark-on volume 120 μ L), by 6 parts of test samples of method parallel processing of embodiment 1 step (1), by embodiment 2 step (3)
Chromatographic condition sample introduction 3 μ L respectively, measures, calculates average recovery, the results are shown in Table 14.
The average recovery test knot of index components ellagic acid and Kaempferol-O-rutinoside in table 14 Chinese medicine Fructus Rubi
Really (n=6)
Embodiment 3
(1) preparation of need testing solution
Chinese medicine Fructus Rubi to 7 places of production is cooked following process respectively:
Chinese medicine Fructus Rubi is pulverized, crosses 40 mesh sieves, accurately weighed 1.0g, put in conical flask, accurate addition 70% methanol
50mL, close plug, weigh 44.6g, room temperature supersound process 30min, weighs, and supplies the weight of loss with 70% methanol aqueous solution, shakes
Even, centrifugal 14000 turns of 10min, take supernatant 1mL.
Each process is repeated 3 times.
(2) preparation of reference substance solution
Accurately weighed adenosine, gallic acid, brevifolin carboxylic acid, progallin A, Kaempferol-O-6-O-.alpha.-L-rhamnosyl-D-glucose. respectively
Glycosides, Kampferol-3-O-glucoside and tiliroside reference substance 5.80mg, 6.90mg, 7.43mg, 6.89mg, 6.33mg, 7.65mg
And 5.89mg, add 100% methanol (containing a small amount of DMSO) respectively and dissolve and be settled to 5mL, accurately weighed ellagic acid reference substance respectively
2.91mg, adds 100% methanol and dissolves and be settled to 10mL, be configured to 1.16mg mL respectively-1Adenosine, 1.38mg mL-1No food
Son acid, 1.49mg mL-1Brevifolin carboxylic acid, 1.38mg mL-1Progallin A, 0.29mg mL-1Ellagic acid,
1.27mg·mL-1Kaempferol-O-rutinoside, 1.53mg mL-1Kampferol-3-O-glucoside, 1.18mg mL-1Hibiscus syriacus
The reference substance storing solution of tree glycosides.
The most accurate absorption adenosine, gallic acid, brevifolin carboxylic acid, progallin A, ellagic acid, Kaempferol-
O-rutinoside, Kampferol-3-O-glucoside and tiliroside reference substance storing solution 200 μ L, 150 μ L, 200 μ L, 400 μ L,
4000 μ L, 500 μ L, 150 μ L and 300 μ L mixing, and by 85% methanol constant volume to 10mL, it is configured to 23.20 μ g mL-1Adenosine,
20.70μg·mL-1Gallic acid, 29.72 μ g mL-1Brevifolin carboxylic acid, 55.12 μ g mL-1Progallin A,
116.40μg·mL-1Ellagic acid, 63.30 μ g mL-1Kaempferol-O-rutinoside, 22.95 μ g mL-1Kaempferol-O-
Glucoside, 35.34 μ g mL-1Tiliroside mixing reference substance solution, and with 1mL volume fraction be 100% methanol solution step by step
Dilution, adenosine concentration is respectively 11.60,5.80,2.90,1.45,0.73,0.36 μ g mL-1, gallic acid concentration is respectively
10.35、5.18、2.59、1.29、0.65、0.32μg·mL-1, brevifolin acid concentration is respectively 14.86,7.43,3.72,
1.86、0.93、0.46μg·mL-1, progallin A is respectively 27.56,13.78,6.89,3.45,1.72,0.86 μ g
mL-1, ellagic acid concentration is respectively 58.20,29.10,14.55,7.28,3.64,1.82 μ g mL-1, Kaempferol-O-Folium Symplocoris Caudatae
Glycoside concentration is respectively 31.65,15.83,7.91,3.96,1.98,0.99 μ g mL-1, Kampferol-3-O-glucoside concentration
It is respectively 11.48,5.74,2.87,1.43,0.72,0.36 μ g mL-1, tiliroside concentration is respectively 17.67,8.84,4.42,
2.21、1.10、0.55μg·mL-1。
(3) Ultra Performance Liquid Chromatography detection
Chromatographic condition: fixing phase: ACQUITY BEH C18 (3.0mm × 100mm, 1.7 μm) chromatographic column；Flowing phase: A phase:
Acetonitrile and B phase: the ratio of 0.1% formic acid is acetonitrile: 0.1% formic acid ratio is 0～2min, 3:97～5:95；2～8min, 5:95
～11.5:88.5；8～13.5min, 11.5:88.5～15.5:84.5；13.5～15min, 15.5:84.5～18:82；15～
18min, 18:82～20:80；18～23min, 20:80～38:62；23～25min, 38:62～70:30；Flow velocity: 0.4mL
min-1；Column temperature 30 DEG C；PDA detector, detection wavelength is 256nm, 265nm, 271nm, 344nm.
Precision draws need testing solution and the mixing reference substance solution 3 μ L of variable concentrations respectively, injects ultra high efficiency liquid phase color
Spectrometer (UPLC) detects.Adenosine, gallic acid, brevifolin carboxylic acid, progallin A, ellagic acid, Kaempferol-
O-rutinoside, Kampferol-3-O-glucoside and tiliroside, the chromatogram of 8 kinds of reference substance solution is respectively such as Fig. 3 A～3D institute
Show.The chromatogram of need testing solution is as a example by the chromatogram (Fig. 3 E～3H) of the Chinese medicine Fructus Rubi need testing solution in the place of production, Zhejiang.
From Fig. 3 A it can be seen that under 256nm wavelength, the retention time of adenosine is 2.16min, during the reservation of ellagic acid
Between be 14.39min.Correspondingly, in Fig. 3 E, the chromatographic peak at 2.16min and 14.39min is respectively the Chinese medicine in the place of production, Zhejiang
Adenosine and the chromatographic peak of ellagic acid in Fructus Rubi need testing solution, and realize good separation with other compositions.From Fig. 3 B permissible
Finding out, under 265nm wavelength, the retention time of tiliroside is 22.12min.Correspondingly, the color in Fig. 3 F, at 22.12min
Spectral peak be exactly the place of production, Zhejiang Chinese medicine Fructus Rubi need testing solution in the chromatographic peak of tiliroside, and realize good point with other compositions
From.From Fig. 3 C it can be seen that under 271nm wavelength, the retention time of gallic acid is 2.67min, progallin A
Retention time is 12.44min.Correspondingly, in Fig. 3 G, the chromatographic peak at 2.67min and 12.44min is respectively the place of production, Zhejiang
Chinese medicine Fructus Rubi need testing solution in gallic acid and the chromatographic peak of progallin A, and realize good point with other compositions
From.From Fig. 3 D it can be seen that under 344nm wavelength, the retention time of brevifolin carboxylic acid is 8.54min, Kaempferol-O-
The retention time of rutinoside is 17.16min, and the retention time of Kampferol-3-O-glucoside is 17.77min.Correspondingly,
In Fig. 3 H, the chromatographic peak at 8.54min, 17.16min and 17.77min is respectively the Chinese medicine Fructus Rubi test sample in the place of production, Zhejiang
Brevifolin carboxylic acid, Kaempferol-O-rutinoside and the chromatographic peak of Kampferol-3-O-glucoside in solution, and and other
Composition realizes good separation.
(4) adenosine, gallic acid, brevifolin carboxylic acid, progallin A, ellagic acid, Kaempferol-O-are set up respectively
The standard curve of rutinoside, Kampferol-3-O-glucoside and tiliroside.
Respectively with adenosine, gallic acid, brevifolin carboxylic acid, progallin A, ellagic acid, Rhizoma Kaempferiae in mixing reference substance
The concentration of phenol-3-O-rutinoside, Kampferol-3-O-glucoside and tiliroside is abscissa, molten with mixing reference substance respectively
Adenosine in the Ultra Performance Liquid Chromatography figure of liquid, gallic acid, brevifolin carboxylic acid, progallin A, ellagic acid, nimbecetin-
The chromatographic peak peak area of 3-O-rutinoside, Kampferol-3-O-glucoside and tiliroside is vertical coordinate, sets up adenosine, no food
Son acid, brevifolin carboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside
Standard curve respective with tiliroside.All with signal to noise ratio (S/N) 10 as quantitative limit, the results are shown in Table 15.As can be known from Table 15, gland
Glycosides, gallic acid, brevifolin carboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kaempferol-O-
Glucoside and tiliroside are good in respective concentration range internal linear relation.
The equation of linear regression of 8 kinds of reference substances, correlation coefficient, the range of linearity, quantitative limit in table 15 Chinese medicine Fructus Rubi
(5) adenosine, gallic acid, brevifolin carboxylic acid, progallin A, ellagic acid, Kaempferol-O-Folium Symplocoris Caudatae are determined
Glucosides, Kampferol-3-O-glucoside and tiliroside be respective content in test sample
Respectively according to adenosine, gallic acid, brevifolin carboxylic acid, Galla Turcica (Galla Helepensis) in need testing solution Ultra Performance Liquid Chromatography figure
Acetoacetic ester, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and the chromatographic peak peak area of tiliroside,
And corresponding standard curve, calculate respectively adenosine, gallic acid, brevifolin carboxylic acid, progallin A, ellagic acid,
Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and the concentration of tiliroside, and calculate confession according to the following formula
The content of 8 kinds of index components in test product:
Content=CDense*V/M。
Obtain the content of 8 kinds of index components in the Chinese medicine Fructus Rubi in 7 places of production according to this formula, the results are shown in Table 16.
8 kinds of index components assay result (Mean ± SD, n=3) in table 16 Chinese medicine Fructus Rubi
Precision, stability, repeatability and average recovery is carried out below as a example by the Chinese medicine Fructus Rubi as Zhejiang of the place of production
Test.
(6) precision test
The accurately weighed Chinese medicine Fructus Rubi 1.0g pulverizing 40 mesh, prepares test sample by the method for embodiment 1 step (1) molten
Liquid, by embodiment 3 step (3) chromatographic condition, continuous sample introduction 6 times, each 3 μ L, 8 kinds of index components in detection need testing solution
Content, evaluates the withinday precision of 8 kinds of index components respectively, the results are shown in Table 17.Test sample is prepared by embodiment 3 step (1)
Solution, by embodiment 3 step (3) chromatographic condition, continuous three days sample introductions, 6 times/day, 3 μ L/ time, measure, evaluate 8 kinds of indexs and become
The day to day precision divided, the results are shown in Table 18.Knowable to table 17 and 18, adenosine that withinday precision test obtains, gallic acid, short
Leaf Lignum Sappan phenolic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tilia
The RSD of glycosides content is respectively 0.68%, 1.74%, 1.53%, 2.45%, 1.43%, 0.58%, 0.58%, 0.90%, in the daytime
The adenosine of precision test acquisition, gallic acid, brevifolin carboxylic acid, progallin A, ellagic acid, Kaempferol-O-rue
The RSD of fragrant glucosides, Kampferol-3-O-glucoside and tiliroside content is respectively 0.97%, 1.49%, 1.98%, 2.78%,
1.61%, 0.68%, 1.46%, 0.94%.Show adenosine, gallic acid, brevifolin carboxylic acid, progallin A, tan flower
The precision of acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside is good, meets assay
Requirement.
The withinday precision result of the test (mg/g, n=6) of 17 8 kinds of index components of table
The day to day precision result of the test (mg/g, n=3) of 18 8 kinds of index components of table
(7) stability test
The accurately weighed Chinese medicine Fructus Rubi 1.0g pulverizing 40 mesh, prepares test sample by the method for embodiment 1 step (1) molten
Liquid, by the chromatographic condition of embodiment 3 step (3), respectively at 0,2,4,8,10 and 12h sample introduction 3 μ L, measures, investigates test sample molten
In liquid, the stability of 8 kinds of index components, the results are shown in Table 19.As known from Table 19, adenosine, gallic acid, brevifolin carboxylic acid, no food
The RSD of sub-acetoacetic ester, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside content is respectively
It is 1.00%, 0.96%, 1.01%, 3.00%, 0.58%, 0.89%, 1.65%, 0.68%.Result shows need testing solution
Middle adenosine, gallic acid, brevifolin carboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, nimbecetin-
In 3-O-glucoside and tiliroside 12h at ambient temperature basicly stable.
The stability test result (mg/g, n=6) of 19 8 kinds of index components of table
(8) replica test
The accurately weighed Chinese medicine Fructus Rubi 0.5g pulverizing 40 mesh, by the method parallel processing 6 parts of embodiment 1 step (1)
Need testing solution, by the chromatographic condition of embodiment 3 step (3) sample introduction 3 μ L respectively, measures, the results are shown in Table 20, adenosine, Galla Turcica (Galla Helepensis)
Acid, brevifolin carboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and
The RSD of tiliroside content is respectively 1.75%, 1.69%, 1.04%, 2.60%, 1.68%, 0.96%, 1.67%, 3.65%.
Result shows that the detection method repeatability of the present invention is good.
The reperformance test result (mg/g, n=6) of 20 8 kinds of index components of table
(9) average recovery test
The accurately weighed Chinese medicine Fructus Rubi 0.5g pulverizing 40 mesh, the most accurately adds and adenosine, Galla Turcica (Galla Helepensis) in test sample
Acid, brevifolin carboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and
Each reference substance storing solution that tiliroside amount is suitable or reference substance solution (adenosine reference substance solution concentration 116 μ g mL-1, add standard type
Long-pending 500 μ L；Gallic acid reference substance solution concentration 138 μ g mL-1, mark-on volume 320 μ L；Brevifolin carboxylic acid reference substance is laid in
Liquid concentration 1486 μ g mL-1, mark-on volume 60 μ L；Progallin A reference substance Stock concentrations 1378 μ g mL-1, mark-on
Volume 120 μ L；Ellagic acid reference substance solution concentration 327 μ g mL-1, mark-on volume 2000 μ L；Kaempferol-O-rutinoside pair
According to product Stock concentrations 1266 μ g mL-1, mark-on volume 150 μ L；Kampferol-3-O-glucoside reference substance solution concentration 153 μ
g·mL-1, mark-on volume 420 μ L；Tiliroside reference substance Stock concentrations 1178 μ g mL-1, mark-on volume 80 μ L), by implementing
6 parts of test samples of the method parallel processing of example 1 step (1), by the chromatographic condition of embodiment 3 step (3) sample introduction 3 μ L respectively, measure,
Calculate average recovery, the results are shown in Table 21.
The average recovery result of the test (n=6) of 8 kinds of index components in table 21 Chinese medicine Fructus Rubi
It should be noted that from embodiment 1,2 and 3 it can be seen that in preparing need testing solution step, difference only exists
Different in the quality of accurately weighed Chinese medicine Fructus Rubi powder, embodiment 1 and 2 is 0.5g, and embodiment 3 is 1.0g.Either
0.5g or 1.0g Chinese medicine Fructus Rubi powder, processes with 50mL 70% methanol, and the addition of 70% methanol is all excessive, all
The purpose fully extracted can be reached.That is embodiment 1,2 is the same with 3 for the preparation method of need testing solution.
Based on this, about test sample in the precision test of each embodiment, stability test, replica test and average recovery test
The preparation method of solution is also the same.
It should be noted that when preparing reference substance solution, embodiment 1 and 2 uses 70% methanol to prepare reference substance solution,
Embodiment 3 uses 85% methanol to prepare reference substance solution.Due in the present invention, when carrying out Ultra Performance Liquid Chromatography detection, sample introduction
Amount is only 3 μ L, and whole detection system Ultra Performance Liquid Chromatography instrument-PDA detector and chromatographic condition are not produced fluctuation, i.e.
The testing result of various index components is not produced impact.
It should be noted that in Fig. 3, by the retention time of 8 kinds of index components in Chinese medicine Fructus Rubi, 1 represents adenosine, 2 generations
Epi-nutgall acid, 3 represent brevifolin carboxylic acid, 4 represent progallin A, 5 represent ellagic acid, 6 represent Kaempferol-O-rue
Fragrant glucosides, 7 represent Kampferol-3-O-glucoside, 8 represent tiliroside, the most as shown in Figure 3A: adenosine (1) and ellagic acid (5)
Reference substance detection wavelength is 256nm；As shown in Figure 3 B: tiliroside (8) reference substance detection wavelength is 265nm；As shown in Figure 3 C: do not have
Gallate-based (2) and progallin A (4) reference substance detection wavelength are 271；As shown in Figure 3 D: brevifolin carboxylic acid (3), Rhizoma Kaempferiae
Phenol-3-O-rutinoside (6), Kampferol-3-O-glucoside (7) reference substance detection wavelength is 344nm.Fig. 3 E and Fig. 3 A couple
Should, adenosine and the chromatographic peak of ellagic acid in detection need testing solution, Fig. 3 F and Fig. 3 B is corresponding, tiliroside in detection need testing solution
Chromatographic peak；Fig. 3 G and Fig. 3 C is corresponding, gallic acid and the chromatographic peak of progallin A in detection need testing solution；Fig. 3 H with
Fig. 3 D is corresponding, brevifolin carboxylic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside in detection need testing solution
Chromatographic peak.
It should be noted that the maximum absorption wavelength scope of ellagic acid is 253～256nm, in embodiment 1 and embodiment 2
Set the detection wavelength of ellagic acid as 253nm.In embodiment 3, a length of 256nm of maximum absorption wave of adenosine, the maximum of the two
Absorbing wavelength difference is little, in order to reduce the number of wavelengths of setting, the detection wavelength of ellagic acid is adjusted to 256nm, to tan flower
The testing result of acid has little to no effect.This is that those skilled in the art can adjust according to practical situation and experience,
Have no effect on the reliability of testing result.
It should be noted that in embodiment 3, when preparing reference substance mixed solution, add a small amount of dimethyl sulfoxide
(DMSO) it is to make the reference substance of 8 kinds of index components fully be thoroughly dissolved in 85% methanol, to various index components
Content detection result does not produce impact.
Though it should be noted that the present invention is to detect many indexes composition simultaneously, but the Ultra Performance Liquid Chromatography used
Instrument is to publish picture respectively according to the detection wavelength set.As a example by embodiment 2, set 2 detection wavelength, respectively 253nm,
265nm, then obtain under 253nm wavelength, reference substance and each figure of test sample, under 265nm wavelength, and reference substance and test sample each
Zhang Tu, altogether 4 figures.
According to the technical scheme of embodiment 3, those skilled in the art are without creative work, permissible according to this technical scheme
Detect adenosine, gallic acid, brevifolin carboxylic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, mountain simultaneously
One or more in naphthol-3-O-glucoside and tiliroside, say, that detect Chinese medicine Fructus Rubi middle finger according to the present invention
The technical scheme of mark composition, detects the guarantor coming under the present invention to the index components in any one or more Chinese medicine Fructus Rubi
Protect scope.
It can be seen from the results above that the present invention utilizes Ultra Performance Liquid Chromatography instrument to measure constructed Chinese medicine Fructus Rubi middle finger
The quantitative analysis method of mark composition, at most can detect adenosine, gallic acid, brevifolin carboxylic acid, progallin A, tan simultaneously
Flower acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside and tiliroside, 8 kinds of index components.The method relative to
The method detected respectively for different index components, shortens detection total time, saves actual cost.And testing result is accurate,
Precision is high, reproducible, and stability is high, and specificity is strong.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit protection scope of the present invention.All
Any modification, equivalent substitution and improvement etc. made within the spirit and principles in the present invention, are all contained in protection scope of the present invention
In.
Claims (5)
1. the assay method of Chinese medicine Fructus Rubi middle finger index composition content, comprises the following steps:
(1) need testing solution is prepared
By Chinese medicine Fructus Rubi that quality is M and V volume 0～100% methanol mixed, obtain the two mixed liquor, determining mixed liquor
Weight M1After, to its carry out extract operation, then with 0～100% methanol the weight of described mixed liquor is complemented to M1, take
Clear liquid, obtains need testing solution；
(2) preparation mixing reference substance solution
Weigh the reference substance of N kind index components in Chinese medicine Fructus Rubi respectively, respectively with 0～100% methanol dissolve, constant volume, obtain
There is the reference substance solution of each index components of concentration known；Wherein, described index components is adenosine, gallic acid, short leaf Soviet Union
Wood phenolic acid, progallin A, ellagic acid, Kaempferol-O-rutinoside, Kampferol-3-O-glucoside, tiliroside, N=
8；
Measure the reference substance solution of each index components the most respectively, mixing, with 0～100% methanol constant volume, obtain mixing reference substance and store up
Standby liquid, in described mixing reference substance storing solution, each index components has concentration known；
By reference substance mixing storing solution with 0～100% methanol stepwise dilution become a series of and be respectively provided with the mixed of different concentration known
Close reference substance solution；
(3) Ultra Performance Liquid Chromatography detection
Chromatographic condition includes: fixing phase: use the chromatographic column of octadecylsilane chemically bonded silica；Flowing phase: acetonitrile: 0.1% first
Acid ratio is 0～2min, 3:97～5:95；2～8min, 5:95～11.5:88.5；8～13.5min, 11.5:88.5～15.5:
84.5；13.5～15min, 15.5:84.5～18:82；15～18min, 18:82～20:80；18～23min, 20:80～38:
62；23～25min, 38:62～70:30；Flow velocity is 0.2～1.0mL/min, and column temperature is 30 DEG C, PDA detector, detects wavelength
For 256nm, 265nm, 271nm and 344nm；
Under described chromatographic condition, by V1The need testing solution of volume and the mixing reference substance solution of each concentration are injected separately into superelevation
In effect liquid phase chromatogram instrument, it is thus achieved that the Ultra Performance Liquid Chromatography figure of need testing solution and the ultra high efficiency of each concentration mixing reference substance solution
Liquid chromatogram；
(4) the N respective standard curve of kind index components is set up
In the Ultra Performance Liquid Chromatography figure of each mixing reference substance solution the chromatographic peak peak area of each index components as vertical coordinate or
Abscissa, to mix in reference substance solution the concentration of each index components as abscissa or vertical coordinate, sets up each index components respectively
Standard curve；
(5) N kind index components respective content in test sample is determined
According to N kind index components respective chromatographic peak peak area in need testing solution Ultra Performance Liquid Chromatography figure, and set up
The standard curve of each index components, calculates the N respective concentration C of kind index components respectivelyDense, and calculate the most respectively
Go out the respective content of N kind index components in test sample:
Content=CDense*V/M。
2. the assay method of Chinese medicine Fructus Rubi middle finger index composition content as claimed in claim 1, it is characterised in that described extraction
For supersound extraction.
3. the assay method of Chinese medicine Fructus Rubi middle finger index composition content as claimed in claim 1, it is characterised in that by described
The weight of mixed liquor complements to M1After, centrifugal, then take supernatant.
4. the assay method of Chinese medicine Fructus Rubi middle finger index composition content as claimed in claim 1, it is characterised in that described 0～
100% methanol is 70～85% methanol.
5. the assay method of Chinese medicine Fructus Rubi middle finger index composition content as claimed in claim 1, it is characterised in that described flow velocity
For 0.4mL/min.
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