CN109490446A - Method that is a kind of while measuring 5 kinds of flavone compounds in ground centipede medicinal material - Google Patents
Method that is a kind of while measuring 5 kinds of flavone compounds in ground centipede medicinal material Download PDFInfo
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Abstract
The invention discloses a kind of methods for measuring 5 kinds of flavonoid contents in ground centipede medicinal material simultaneously, comprising: the preparation of test solution, the preparation of reference substance solution and high performance liquid chromatography detection analysis and etc..The present invention detects 5 kinds of flavones active components, i.e. afzelechin -3-O- α-L- rhamnose, astragalin, Multiflorin B, afzclin and Multiflorin A in ground centipede medicinal material simultaneously for the first time.Detection method high sensitivity of the invention, reproducible, the rate of recovery is high.The present invention establishes the quality controling research using main chemical compositions in ground centipede as the detection method of the ground centipede medicinal material of Testing index, for ground centipede medicinal material and clinical application is instructed to be of great significance.
Description
Technical field
The invention belongs to detection technique fields, and in particular to a kind of to measure 5 kinds of flavonoids in ground centipede medicinal material simultaneously
The method of object.
Background technique
Ground centipede derives from the more plumage Rhizoma Arthromeritis maireis of Plants of PolypodiaceaeArthromeris mairei(Brause) Ching.Not
Name Chinese atropanthe root, Cortex Ampelopsis Aconitifoliae Radicis etc., are Yi nationality of Yunnan conventional crude drugs, and Yi nationality's medicine is entitled " I carries on the back promise ", and free translation is " medicine that belly is swollen ".Its taste
Hardship, cold nature, returns spleen, stomach meridian, have it is active, remove food retention, defaecation, fall fire function, for treating dyspepsia stomachache, abdominal distension, constipation, wind
Wet arthralgia and myalgia, sciatica, headache, toothache.The plant is mainly distributed on the ground such as Yunnan, Tibet, Sichuan, Burma and India
The north also has.
Ground centipede medicinal material is one of preparation " gut purge Tongbian capsule " prescription medicine, is recorded at present in " Yunnan Province's medicinal material mark
It is quasi- " (one) (2005 editions).4 flavonoid glycoside compounds are isolated from ground centipede currently, having reported, such as: multiflorin, kaempferia galamga
Element -3-O- β-D- glucopyranose (1 → 4)-α-L- rhamnopyranosyloxyhy glucosides, arthromerin A, arthromerin B.Invention
People in the course of the research, isolated 5 flavone compounds from ground centipede medicinal material for the first time: Multiflorin A,
Multiflorin B, astragalin, afzclin and afzelechin -3-O- α-L- rhamnose, wherein Multiflorin A,
Multiflorin B, afzelechin -3-O- α-L- rhamnose are its main components, astragalin and afzclin be have compared with
Johnson & Johnson manages active flavonoid glycoside ingredient.This 5 flavonoid glycoside ingredients are the active constituents of ground centipede medicinal material, are established with this 5
Flavonoid glycoside ingredient is the ground centipede medicinal material detection method of Testing index, and the quality of ground centipede medicinal material and related preparations is controlled
It is of great significance.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for measuring 5 kinds of flavone compounds in ground centipede medicinal material simultaneously.
The object of the invention, including test solution preparation, reference substance solution preparation, inspection is realized by the following method in the present invention
Survey and analytical procedure, specifically include
(1) preparation of test solution: taking about 1 g of ground centipede medicinal powder, accurately weighed, sets in conical flask, first is added in precision
50 mL of alcohol water, weighed quality are heated to reflux；It lets cool, then weighed weight, solubilizer is supplied the weight of less loss, shaken up, filtration；Essence
5 mL of close measurement filtrate is set in the volumetric flask of 25 mL, is added methanol-water constant volume, is shaken up to get test solution；
(2) prepared by reference substance solution:
A, accurately weighed afzelechin -3-O- α-L- rhamnose reference substance, being configured to concentration with 50% methanol is 20 μ g mL-1's
Reference substance solution, it is spare through 0.45 μm of filtering with microporous membrane to get reference substance solution A；
B, the accurately weighed astragalin of difference, Multiflorin B, afzclin, Multiflorin A reference substance, after mixing
0.8 μ g mL of astragalin concentration is configured to methanol-1, 54 μ g mL of Multiflorin B concentration-1, afzclin concentration
1.5 μg•mL-1, 20 μ g mL of Multiflorin A concentration-1Mixed solution, obtain reference substance solution up to reference substance solution
B, it is spare；
(3) it detects: test solution, reference substance solution A and reference substance solution B injection high performance liquid chromatograph is detected；
(4) quantitative analysis of 5 kinds of flavonoid components in ground centipede medicinal material is carried out: with external standard method with the flavones of known concentration
The chromatographic peak area of class compound compares its respective concentration and carries out regression analysis, obtains standard curve, carries out to test solution
Measurement measures the chromatographic peak area of 5 kinds of flavonoid components in detection test solution, substitutes into standard curve, acquire ground
The content of 5 kinds of flavonoid components in centipede medicinal material.
The granularity of ground as described in step (1) centipede medicinal powder is 65 mesh.
Methanol water concentration as described in step (1) is 75% methanol.
It is as described in step (1) to be heated to reflux the time as 1 h.
Extracting temperature as described in step (1) is 70 DEG C ± 2 DEG C.
The High Performance Liquid Chromatography condition of test solution described in step (3) are as follows:
A, chromatographic column: Waters C18(4.6 nm × 250 mm, 5 μm), mobile phase are as follows: V (methanol): V (water)=35:65 is isocratic
Elution；Flow velocity is 1.0 mLmin -1, column temperature is 25 DEG C, and Detection wavelength is 230 nm, 10 μ L of sample volume；
B, chromatographic column: Waters SunFire C18(4.6 × 250 mm, 5 μm), mobile phase are 0. 2% phosphate aqueous solution
(C)-methanol (D), gradient elution: 0~30 min, C:D=53: 47；30~40 min, 47% D → 66% D；Flow velocity
1.0 mL·min -1, 265 nm of Detection wavelength, 25 DEG C of column temperature, 10 μ L of sample volume；
The High Performance Liquid Chromatography condition of reference substance solution A described in step (3) are as follows:
Chromatographic column: Waters C18(4.6 nm × 250 mm, 5 μm), mobile phase are as follows: V (methanol): V (water)=35:65 is isocratic washes
It is de-；Flow velocity is 1.0 mLmin -1, column temperature is 25 DEG C, and Detection wavelength is 230 nm, 10 μ L of sample volume.
The High Performance Liquid Chromatography condition of reference substance solution B described in step (3) are as follows:
Chromatographic column: Waters SunFire C18(4.6 × 250 mm, 5 μm), mobile phase are 0. 2% phosphate aqueous solution
(C)-methanol (D), gradient elution: 0~30 min, C:D=53: 47；30~40 min, 47% D → 66% D；Flow velocity
1.0 mL·min -1, 265 nm of Detection wavelength, 25 DEG C of column temperature, 10 μ L of sample volume.
The present invention clearly contains Multiflorin A, Multiflorin B, afzelechin -3- in centipede medicinal material for the first time
This 5 flavone compounds of O- α-L- rhamnose, astragalin and afzclin, and using this 5 compounds as Testing index, point
The method for not establishing measurement afzelechin -3-O- α-L- rhamnose, and measure simultaneously Multiflorin A,
The method of Multiflorin B, astragalin and afzclin grind the quality control of ground centipede medicinal material and its related preparations
Study carefully and clinical application is instructed to be of great significance.
Detailed description of the invention
Fig. 1 is 5 flavone compound structural formulas
Wherein: 1, afzelechin -3-O- α-L- rhamnose；2, astragalin；3,Multiflorin B；4, afzclin；5,
Multiflorin A。
Fig. 2 is the high-efficient liquid phase chromatogram of afzelechin -3-O- α-L- rhamnose reference substance solution
Wherein: 1, afzelechin -3-O- α-L- rhamnose.
Fig. 3 be ground centipede medicinal material sample solution high-efficient liquid phase chromatogram (measurement afzelechin -3-O- α-L- rhamnose,
Chromatographic condition A)
Wherein: 1, afzelechin -3-O- α-L- rhamnose.
Fig. 4 is afzelechin -3-O- α-L- rhamnose standard curve.
Fig. 5 be afzelechin -3-O- α-L- rhamnose nuclear magnetic resonance spectroscopy (1H NMR) figure.
Fig. 6 be afzelechin -3-O- α-L- rhamnose carbon-13 nmr spectra (13C NMR) figure.
Fig. 7 be Multiflorin A, Multiflorin B, astragalin and afzclin mixed reference substance solution height
Effect liquid phase chromatogram figure
Wherein: 1, astragalin；2,Multiflorin B；3, afzclin；4,Multiflorin A.
Fig. 8 be ground centipede medicinal material sample solution high-efficient liquid phase chromatogram (while measure Multiflorin A,
Multiflorin B, astragalin and afzclin, chromatographic condition B)
Wherein: 1, astragalin；2,Multiflorin B；3, afzclin；4,Multiflorin A.
Fig. 9 be astragalin nuclear magnetic resonance spectroscopy (1H NMR) figure.
Figure 10 be astragalin carbon-13 nmr spectra (13C NMR) figure.
Figure 11 be Multiflorin B nuclear magnetic resonance spectroscopy (1H NMR) figure.
Figure 12 be Multiflorin B carbon-13 nmr spectra (13C NMR) figure.
Figure 13 be afzclin nuclear magnetic resonance spectroscopy (1H NMR) figure.
Figure 14 be afzclin carbon-13 nmr spectra (13C NMR) figure.
Figure 15 be Multiflorin A nuclear magnetic resonance spectroscopy (1H NMR) figure.
Figure 16 be Multiflorin A carbon-13 nmr spectra (13C NMR) figure.
Specific embodiment
The method of the present invention is described in further detail below by embodiment, but protection scope of the present invention is not limited to
The content is in embodiment unless otherwise specified conventional commercial reagent using reagent, or reagent obtained according to a conventional method.
The present invention relates to a kind of method for measuring 5 kinds of flavone compounds in ground centipede medicinal material simultaneously is real by the following method
Existing the object of the invention, including test solution preparation, reference substance solution preparation, detection and analysis step, specifically include
(1) preparation of test solution: taking about 1 g of ground centipede medicinal powder, accurately weighed, sets in conical flask, first is added in precision
50 mL of alcohol water, weighed quality are heated to reflux；It lets cool, then weighed weight, solubilizer is supplied the weight of less loss, shaken up, filtration；Essence
5 mL of close measurement filtrate is set in the volumetric flask of 25 mL, is added methanol-water constant volume, is shaken up to get test solution.
Ground centipede medicinal material is clayed into power, and the methanol-water that mass volume ratio is 1g:50mL is added and is heated to reflux so that 5 kinds of Huangs
Ketone compounds can be dissolved out sufficiently from ground centipede medicinal material；The weighed quality after methanol-water is added, and put being heated to reflux
Solubilizer supplies the weight of less loss after cold, it is therefore intended that so that sample size measurement is accurate.
Preferably, the granularity of ground centipede medicinal powder is 65 mesh, methanol water concentration is 75% methanol, to be heated to reflux the time be 1
H, Extracting temperature is 70 DEG C ± 2 DEG C.
(2) prepared by reference substance solution:
A, accurately weighed afzelechin -3-O- α-L- rhamnose reference substance, being configured to concentration with 50% methanol is 20 μ g mL-1's
Reference substance solution, it is spare through 0.45 μm of filtering with microporous membrane to get reference substance solution A；
B, the accurately weighed astragalin of difference, Multiflorin B, afzclin, Multiflorin A reference substance, after mixing
0.8 μ g mL of astragalin concentration is configured to methanol-1, 54 μ g mL of Multiflorin B concentration-1, afzclin concentration
1.5 μg•mL-1, 20 μ g mL of Multiflorin A concentration-1Mixed solution, obtain reference substance solution up to reference substance solution
B, it is spare；
(3) it detects: test solution, reference substance solution A and reference substance solution B injection high performance liquid chromatograph is detected；
Test sample is detected twice, and chromatographic condition is respectively as follows:
A, chromatographic column: Waters C18(4.6 nm × 250 mm, 5 μm), mobile phase are as follows: V (methanol): V (water)=35:65 is isocratic
Elution；Flow velocity is 1.0 mLmin -1, column temperature is 25 DEG C, and Detection wavelength is 230 nm, 10 μ L of sample volume；
B, chromatographic column: Waters SunFire C18(4.6 × 250 mm, 5 μm), mobile phase are 0. 2% phosphate aqueous solution
(C)-methanol (D), gradient elution: 0~30 min, C:D=53: 47；30~40 min, 47% D → 66% D；Flow velocity
1.0 mL·min -1, 265 nm of Detection wavelength, 25 DEG C of column temperature, 10 μ L of sample volume；
The chromatographic condition of reference substance solution A is A, and the chromatographic condition of reference substance solution B is A.
(4) quantitative analysis of 5 kinds of flavonoid components in ground centipede medicinal material is carried out: with external standard method with known concentration
The chromatographic peak area of flavone compound compares its respective concentration and carries out regression analysis, standard curve is obtained, to test solution
It is measured, measures the chromatographic peak area of 5 kinds of flavonoid components in detection test solution, substitute into standard curve, ask
Obtain the content of 5 kinds of flavonoid components in ground centipede medicinal material.
Test solution of the invention prepares simplicity, and consumption of organic solvent is few；Detection method high sensitivity, it is reproducible,
The rate of recovery is high.The present invention specifies for the first time contains Multiflorin A, Multiflorin B, afzelechin-in centipede medicinal material
This 5 flavone compounds of 3-O- α-L- rhamnose, astragalin and afzclin, and using this 5 compounds as Testing index,
The method for establishing measurement afzelechin -3-O- α-L- rhamnose respectively, and measure simultaneously Multiflorin A,
The method of Multiflorin B, astragalin and afzclin grind the quality control of ground centipede medicinal material and its related preparations
Study carefully and is of great significance.
Embodiment 1: the measurement of afzelechin -3-O- α-L- rhamnose in ground centipede medicinal material
1, instrument and material
High performance liquid chromatograph (Agilent:1260) is equipped with quaternary pump, autosampler, column oven, UV detector, electronic
Pulverizer, medicine sieve (No. 4 sieves, 65 mesh), electronic balance (AUW120D, SHIMADZU), excellent general serial Superpure water machine (UPC-
I10T), volumetric flask (50 mL, 25 mL), band plug conical flask (50 mL).
Afzelechin -3-O- α-L- rhamnose reference substance is isolated from ground centipede medicinal material by this laboratory, through efficient
Liquid-phase chromatographic analysis (Detection wavelength 230nm), purity 98.23%；Ground centipede medicinal material is purchased from market, through in Yunnan University of Traditional Chinese Medicine
Medicine identification teaching and research room associate professor Yang Zhuya is accredited as ground centipedeArthromeris mairei(Brause) dry root of Ching
Stem.
Water, methanol；For the reagent methanol (chromatographically pure Merck S. A.) of HPLC detection, water is ultrapure water；It is real
Testing using water is pure water, methanol (the analysis Tianjin net income An Longbohua medical chemistry Co., Ltd).
2, experimental method
2.1 chromatographic condition
Chromatographic column: Waters C18(4.6 nm × 250 mm, 5 μm), mobile phase are as follows: V (methanol): V (water)=35:65 is isocratic washes
It is de-；Flow velocity: 1.0mL/min；Column temperature: 25 DEG C；Detection wavelength: 230 nm；10 μ L of sample volume.Under this chromatographic condition, A Fu beans
The chromatographic peak of element -3-O- α-L- rhamnose is preferably separated with other components peak, as a result sees Fig. 2,3.
The preparation of 2.2 reference substance solutions
Accurately weighed 2.5 mg of afzelechin -3-O- α-L- rhamnose reference substance, is placed in 25 mL volumetric flasks, and 50% methanol is added
Dissolution, constant volume shake up.Precision draws 5 mL constant volume liquid in 25 mL volumetric flasks, with 50 % methanol constant volumes, shakes up.It is configured to dense
Degree is 20 μ g mL-1Reference substance solution, it is spare through 0.45 μm of filtering with microporous membrane.
The preparation of 2.3 test solutions
Precision weighs 1 g medicinal powder, accurately weighed, sets in conical flask, and 75 % methanol, 50 mL, weighing is added.It is heated to reflux and mentions
60 min are taken, Extracting temperature is 70 DEG C ± 2 DEG C.It is cooled to room temperature, weighs.The weight of less loss is supplied with 75 % methanol.Filtering, essence
Close 5 mL of measurement subsequent filtrate is set in 25 mL volumetric flasks, and 75 % methanol constant volumes are added to graduation mark, shakes up, is filtered with 0.45 μm of micropore
Film filtering, it is spare.
3, methodological study
3.1 standard curves and linear relationship are investigated
Accurate 1,5,10,20, the 50 μ L of afzelechin -3-O- α-L- rhamnose reference substance test solution that draws distinguishes sample introduction respectively,
It is analyzed by " 2.1 " item chromatographic condition, measures peak area.Measurement result is as shown in table 1:
1 afzelechin -3-O- α-L- rhamnose linear relationship of table investigates result
Using peak area as Y-axis ordinate, afzelechin -3-O- α-L- rhamnose sample volume (μ g) is X-axis abscissa, is returned
Equation: Y=5022.6X -25.944 (r=0.99995) shows afzelechin -3-O- α-L- rhamnose 0.02104
The good (see figure 4) of linear relationship within the scope of~1.05200 μ g.
3.2 Precision Experiment
Precision draws 10 μ L of afzelechin -3-O- α-L- rhamnose reference substance solution, repeats sample introduction 6 times, presses " 2.1 " item chromatography
Sample introduction is analyzed for condition, and corresponding peak area is as shown in table 2.Calculate afzelechin -3-O- α-L- rhamnose average peak area
Are as follows: 938.633.RSD value are as follows: 0.42034 %.Show that instrument precision is good.
2 afzelechin -3-O- α-L- rhamnose precision of table investigates measurement result
3.3 stability experiment
According to " 2.3 " item preparation method prepare 1 part of test solution, respectively 0 after test solution prepares, 2,4,8,12,
Sample introduction is analyzed by the chromatographic condition of " 3.1.2 " by 24 h, the peak area of afzelechin -3-O- α-L- rhamnose in measurement ground centipede,
The results are shown in Table 3.The RSD value of afzelechin -3-O- α-L- rhamnose peak area are as follows: 0.825 %, explanatorily centipede sample is molten
Liquid is good in 24 h internal stabilities.
3 afzelechin -3-O- α-L- rhamnose stability experiment measurement result of table
3.4 repeated experiment
It takes with 6 parts of medicinal powder of centipede of a batch ground, number consecutively.It is prepared respectively according to " 2.3 " item preparation method.Successively exist
Sample introduction is analyzed under the chromatographic condition of " 2.1 ", the peak area of afzelechin -3-O- α-L- rhamnose in measurement ground centipede, according to phase
It closes formula and calculates content.The results are shown in Table 4, afzelechin -3-O- α-L- rhamnose average content are as follows: 0.2999 %, RSD value
Are as follows: 1.742 %.Show this method for extract ground centipede in afzelechin -3-O- α-L- rhamnose it is reproducible.
4 afzelechin -3-O- α-L- rhamnose repetition measurement result of table
The experiment of 3.5 sample recovery rates
Weigh 6 parts of test sample, it is accurately weighed, it sets in conical flask, accurate respectively that afzelechin -3-O- α-L- rhamnose is added is molten
Liquid volatilizes, and by the test solution prepared under " 2.2 " item, 10 μ L of sample introduction, divides according to the chromatographic condition sample introduction of " 2.1 " item respectively
Analysis, measurement peak area calculate the rate of recovery according to related publicity.The results are shown in Table 5.Mean sample recovery rate are as follows: 100.22%,
RSD value is 1.99%.Show that this method accuracy is good.
5 afzelechin -3-O- α-L- rhamnose of table sample-adding recycling measurement result
3.6 sample size measurement results
Take the ground centipede sample of two batches, 3 parts of every batch of.Test solution is prepared according to the preparation method under " 2.3 " item, successively
Precision draws 10 μ L sample introductions, measures in the chromatographic condition sample introduction of " 2.1 " item, measurement result is shown in Table 6.
6 different batches of table centipede afzelechin -3-O- α-L- rhamnose assay result (n=3)
4, the nuclear magnetic data of reference substance afzelechin -3-O- α-L- rhamnose
1H-NMR (MeOD, 500Hz), δ 5.95 (1H, d,J =2.3Hz, H-6), δ 5.84 (1H, d,J =
2.3 Hz, H-8), δ 7.72 (2H, d,J =8.6 Hz, H-2、, H-3、), δ 6.78(2H, d,J = 8.6
Hz, H-2、, H-6、), δ 4.24 (1H, d,J =1.5Hz, H-2), δ 1.23 (2H, d,J =6.25 Hz, H-
4 )；13C-NMR(MeOD, 125Hz), δ 81.13 (C-2), δ 76.20 (C-3), δ 28.2 (C-4), δ 156.9 (
C-5), δ 96.4 (C-6), δ 157.5 (C-7), δ 95.4 (C-8), δ 158.5 (C-9), 100.7 (C- of δ
10), 131.2 (C-1 of δ、), 129.4 (C-2 of δ、), 116.0 (C-3 of δ、), 157.9 (C-4 of δ、), 116.0 (C- of δ
5、), 129.4 (C-6 of δ、)。
Fig. 5 be afzelechin -3-O- α-L- rhamnose nuclear magnetic resonance spectroscopy (1H NMR) figure, Fig. 6 is afzelechin -3-
O- α-L- rhamnose carbon-13 nmr spectra (13C NMR) figure.Nuclear magnetic data proves that the reference substance is afzelechin -3-O- α-L-
Rhamnose.
Embodiment 2: at the same measure ground centipede medicinal material in Multiflorin A, Multiflorin B, astragalin and
Afzclin.
1, instrument and material
High performance liquid chromatograph (Agilent:1260) is equipped with quaternary pump, autosampler, column oven, UV detector, electronic
Pulverizer, medicine sieve (No. 4 sieves, 65 mesh), electronic balance (AUW120D, SHIMADZU), excellent general serial Superpure water machine (UPC-
I10T), volumetric flask (50 mL, 25 mL), band plug conical flask (50 mL).
Multiflorin A, Multiflorin B, astragalin and afzclin reference substance are by this laboratory from ground Wu
It is isolated in centipede medicinal material, through efficient liquid phase chromatographic analysis (Detection wavelength 265nm), purity is respectively 98.11%, 99.02%,
98.65%,98.48%；Ground centipede medicinal material is purchased from market, identifies through Yunnan University of Traditional Chinese Medicine Chinese traditional medicine identification teaching and research room associate professor Yang Zhuya
For ground centipedeArthromeris mairei(Brause) dry rhizome of Ching.
Water, methanol；For the reagent methanol (chromatographically pure Merck S. A.) of HPLC detection, water is ultrapure water, phosphorus
Sour (Guangdong Guanghua Science and Technology Co., Ltd.)；Experiment is pure water, methanol (the analysis Tianjin net income An Longbohua doctor using water
Medicine Chemical Co., Ltd.).
2, experimental method
2.1 chromatographic condition
Waters SunFire C18Chromatographic column (4.6 × 250 mm, 5 μm), mobile phase are 0. 2% phosphate aqueous solution (C)-
Methanol (D), gradient elution (0~30 min, C:D=53: 47；30~40min, 47% D → 66% D)；Flow velocity 1.0
mL·min-1, 265 nm of Detection wavelength, 25 DEG C of column temperature, 10 μ L of sample volume.Under above-mentioned chromatographic condition, each component separating degree is good
Good, mixing reference substance, sample HPLC figure are shown in Fig. 7,8.
The preparation of 2.2 reference substance solutions
Precision weighs astragalin reference substance 5.01mg, is placed in 250 mL measuring bottles, adds methanol to dissolve and is settled to scale, shakes
It is even, as reference substance stock solution；Precision weighs 13.50 mg of Multiflorin B reference substance, is placed in 10 mL measuring bottles, adds first
Alcohol dissolves and is settled to scale, shakes up, as reference substance stock solution；Precision weighs 9.94 mg of afzclin reference substance, is placed in
In 250 mL measuring bottles, adds methanol to dissolve and be settled to scale, shake up, as reference substance stock solution；Precision weighs
5.02 mg of Multiflorin A reference substance, is placed in 10 mL measuring bottles, adds methanol to dissolve and is settled to scale, shakes up, as
Reference substance stock solution；It is accurate respectively to measure above-mentioned each 1ml of reference substance stock solution and set in 25 mL measuring bottles, add methanol dilution to scale,
It shakes up, it is spare.
The preparation of 2.3 test solutions
About 1 g of this product medicinal powder is taken, it is accurately weighed, it sets in stuffed conical flask, 75% methanol-water, 50 mL, weighed matter is added in precision
Amount, is heated to reflux 1 h.It lets cool, then weighed quality, solubilizer is supplied the weight of less loss, shaken up, filtration.Precision measures subsequent filtrate 5
ML is set in the measuring bottle of 25 mL, is added 75% methanol constant volume, is shaken up, spare.
3, methodological study
3.1 system suitability
Precision measures mixed reference substance solution, test solution and each 10 μ L of blank solution, the sample introduction point under above-mentioned chromatographic condition
Analysis.The results show that chromatographic peak identical with retention time of reference substance is presented in test solution chromatography；Astragalin,
Multiflorin B, afzclin, Multiflorin A chromatographic peak adjacent thereto separating degree be all larger than 1.5, tailing factor
0.97~1.10.
3.2 the range of linearity
The mixed reference substance solution for taking series of concentrations obtained under " 2.2 " item, by 1 μ L, 3 μ L, 5 μ L, 10 μ L, 20 μ L, 30 μ L,
40 μ L distinguish sample introduction, and using peak area as ordinate (Y), sample volume is that abscissa (X) draws standard curve, and line of going forward side by side returns
Return, obtain astragalin, Multiflorin B, afzclin, Multiflorin A regression equation.The experimental results showed that respectively
Ingredient is in good linear relationship in respective concentration range, the results are shown in Table 7.
The linear relationship of 7. 4 kinds of flavones ingredients of table investigates result
3.3 Precision Experiment
Precision draws 10 μ L of mixed reference substance solution, continuous sample introduction 6 times, calculates astragalin, Multiflorin B, A Fu
Beans glycosides, Multiflorin A peak area RSD be respectively 1.65%, 0.19%, 1.87%, 0.37%(n=6), show instrument precision
Degree is good.
3.4 stability experiment
Take same test solution, the 10 μ L of sample introduction after 0,2,4,8,12,24 h after preparation, measure astragalin,
Multiflorin B, afzclin, Multiflorin A peak area average value be respectively 30.79,1235.6,53.48,
445.81, RSD values are respectively 1.50%, 0.34%, 1.24%, 0.48%, show having good stability for this method.
3.5 repeated experiment
Precision is weighed with 6 parts of medicinal material of centipede, every part of about 1 g of a batch ground, and it is molten to prepare 6 parts of test samples in parallel by method under " 2.3 " item
Liquid simultaneously measures in accordance with the law.As a result astragalin, the average content difference of Multiflorin B, afzclin, Multiflorin A
For 0.286,11.075,0.238,7.772 mgg-1, RSD value is respectively 2.47%, 1.55%, 2.30%, 1. 84%, shows this
The repeatability of method is good.
The experiment of 3.6 sample recovery rates
Precision weigh known content 0.5 g of sample (every 1 g in centipede medicinal material containing 0.286 mg of astragalin,
11.075 mg of Multiflorin B, 0.238 mg of afzclin, 7.772 mg of Multiflorin A), parallel 6 parts, point
It is inaccurate that astragalin (0.143 mgmL is added-1), Multiflorin B(5.55 mgmL-1), afzclin
(0.120 mgmL mgmL-1), Multiflorin A(3.85 mgmL-1) 1 mL of mixed reference substance solution, volatilize,
By legal system available test sample solution below " 2.3 " item, sample introduction measure and calculation sample recovery rate.As a result astragalin, Multiflorin
B, afzclin, Multiflorin A sample recovery rate be respectively 95.38%, 97.38%, 100.42%, 105.36%, RSD value
Respectively 1.82%, 2.64%, 1.43%, 1.36%, show that the rate of recovery of this method is good, the results are shown in Table 8.
The average recovery rate result (n=6) of 8. 4 kinds of flavones ingredients of table
The measurement of 3.7 sample sizes
The ground centipede medicinal material of 2 different batches is taken respectively, precision weighs 1 g, it is prepared into test solution by " 2.3 " method, then
The sample introduction under the chromatographic condition of " 2.1 " calculates astragalin, Multiflorin B, afzclin, Multiflorin A and contains
Amount, the results are shown in Table 9.
9. different batches of table ground assay result of 4 flavones ingredients in centipede medicinal material
According to chromatograms the result shows that, under such an approach astragalin, Multiflorin B, afzclin,
This 4 ingredients of Multiflorin A can be realized with other compositions preferably to be separated, and the method has accuracy and feasibility.
4, reference substance astragalin, Multiflorin B, afzclin, Multiflorin A nuclear magnetic data
Multiflorin A:1H-NMR (MeOD, 500 MHz), δ 7.7 (2H, d,J=8.8Hz, H-2 ', H-6 '), δ
6.92(2H, d, J=8.8Hz, H-3 ', H-5 '), δ 6.32 (1H, d,J=1.9Hz, H-8), δ 6.15 (1H, d,
J=2.0Hz, H-6), δ 5.33 (1H, s, J=1.5Hz, H-1 ' '), δ 4.47(1H, s, J=7.9Hz H-1 ' ' '), δ
2.0(3H, s, CH3COO), δ 0.92(3H, d,J=1.5Hz H-6 ' ')；13C-NMR ( MeOD,100 MHz ) δ159.3
(C-2), δ 136.1 (C-3), δ 179.5 (C-4), δ 163.1 (C-5), δ 99.9 (C-6), δ 165.8 (C-7), 94.9 (C- of δ
8), δ 158.4 (C-9), δ 105.9 (C-10), δ 122.6 (C-1 '), δ 131.9 (C-2 ', 5 '), δ 116.5 (C-3 ', 5 '), δ
105.6 (C-1 ' ' '), δ 83.2 (C-5 ' ' '), δ 75.2 (C-3 ' ' '), δ 71.8 (C-2 ' ' '), δ 71.7 (C-4 ' ' '), δ 64.8
(C-6 ' ' '), 20.81 (CH of δ3), δ 172.8 (C=O), δ 103.3 (C-1 ' '), δ 78.0 (C-5 ' '), δ 75.9 (C-3 ' '), δ
72.1 (C-2 ' '), δ 70.5 (C-4 ' '), δ 17.8 (C-6 ' ').
Multiflorin B:1H-NMR (MeOD, 500 MHz): δ 12.6 (1H, s, 5-OH), δ 7.75 (2H, d,J
=8.8Hz, H-2 ', 6 '), δ 6.92 (2H, d,J=8.8Hz, H-3 ', 5 '), δ 6.43 (1H, d,J = 1.9Hz,
H-8), δ 6.22 (1H, d,J=2.0Hz, H-6), δ 5.48(1H, s, H-1 ' '), δ 4.05(1H, s, H-1 ' ' '), δ
0.90(3H, d, rha-CH3)；13C-NMR (MeOD, 500 MHz): δ 157.3 (C-2), δ 134.4 (C-3), 177.68 (C- of δ
4), δ 161.3 (C-5), δ 98.8 (C-6), δ 164.3 (C-7), δ 93.81 (C-8), δ 156.5 (C-9), 104.7 (C- of δ
10), δ 120.4 (C-1 '), δ 131.6 (C-2 ', 5 '), δ 115.5 (C-3 ', 5 '), δ 104.1 (C-1 ' ' '), 82.0 (C- of δ
5 ' ' '), δ 74.5 (C-3 ' ' '), δ 69.8 (C-2 ' ' '), δ 69.7 (C-4 ' ' '), δ 60.96 (C-6 ' ' '), 101.8 (C- of δ
1 ' '), δ 76.9 (C-5 ' '), δ 76.6 (C-3 ' '), δ 70.3 (C-2 ' '), δ 68.9 (C-4 ' '), δ 17.3 (C-6 ' ').
Afzclin:1H-NMR (MeOD, 500Hz), δ 6.15 (1H, d,J =2.1 Hz, H-6), δ 6.31
(1H, d,J =2.1 Hz, H-8), δ 7.72(2H, dt,J =6.9,2 Hz, H-2 '、, H-6 '、), δ 6.90
(2H, dt,J =6.9,2 Hz, H-3 '、, H-5 '、), δ 5.36 (1H, d,J =1.65 Hz, H-1 ' '),13C-NMR(MeOD, 125Hz), δ 158.4 (C-2), δ 136.2 (C-3), δ 179.5 (C-4), 163.1 (C-5 of δ
), δ 99.8 (C-6), δ 165.8 (C-7), δ 94.8 (C-8), δ 159.2 (C-9), δ 105.9 (C-10), δ
122.6 ( C-1’、), δ 131.9 (C-2 ', 6 '、), δ 116.5 (C-3 ', 5 '、), 161.5 (C-4 ' of δ、), δ 103.5 (
C1 ' '), δ 72.1 (C2 ' '), δ 72.0 (C3 ' '), δ 73.2 (C4 ' '), δ 71.9 (C5 ' '), δ 17.7 (C6 ' ').
Astragalin:1H-NMR (MeOD, 500Hz), δ 6.19 (1H, d,J =2.1 Hz, H-6), δ 6.39
(1H, d,J =2.1 Hz, H-8), δ 8.04(2H, dt,J =6.9,2 Hz, H-2 '、, H-6 '、), δ 6.87
(2H, dt,J =6.9,2 Hz, H-3 '、, H-5 '、), δ 5.25 (1H, s, H-1 ' '),13C-NMR(MeOD,
125Hz), δ 158.5 (C-2), δ 135.4 (C-3), δ 179.5 (C-4), δ 163.1 (C-5), δ 99.9 (
C-6), δ 166.0 (C-7), δ 94.7 (C-8), δ 159.1 (C-9), δ 105.7 (C-10), 122.8 (C- of δ
1’、), δ 132.3 (C-2 ', 6 '、), δ 116.1 (C-3 ', 5 '、), 161.6 (C-4 ' of δ、), δ 104.0 (C1 ' '), δ
75.7 (C2 ' '), δ 78.4 (C3 ' '), δ 71.3 (C4 ' '), δ 78.0 (C5 ' '), δ 62.6 (C6 ' ').
Fig. 9 be astragalin nuclear magnetic resonance spectroscopy (1H NMR) figure, Figure 10 be astragalin carbon-13 nmr spectra (13C
NMR) figure；Figure 11 be Multiflorin B nuclear magnetic resonance spectroscopy (1H NMR) figure, Figure 12 is the nuclear-magnetism of Multiflorin B
Resonance carbon spectrum (13C NMR) figure；Figure 13 be afzclin nuclear magnetic resonance spectroscopy (1H NMR) figure, Figure 14 is the core of afzclin
Magnetic resonance carbon spectrum (13C NMR) figure；Figure 15 be Multiflorin A nuclear magnetic resonance spectroscopy (1H NMR) figure, Tu16Wei
Multiflorin A carbon-13 nmr spectra (13C NMR) figure.Nuclear magnetic data show compound be respectively astragalin,
Multiflorin B, afzclin, Multiflorin A.
Claims (8)
1. a kind of method for measuring 5 kinds of flavonoid contents in ground centipede medicinal material simultaneously, it is characterised in that including test sample
Solution preparation, reference substance solution preparation, detection and analysis step, specifically include
(1) preparation of test solution: taking about 1 g of ground centipede medicinal powder, accurately weighed, sets in conical flask, first is added in precision
50 mL of alcohol water, weighed quality are heated to reflux；It lets cool, then weighed weight, solubilizer is supplied the weight of less loss, shaken up, filtration；Essence
5 mL of close measurement filtrate is set in the volumetric flask of 25 mL, is added methanol-water constant volume, is shaken up to get test solution；
(2) prepared by reference substance solution:
A, accurately weighed afzelechin -3-O- α-L- rhamnose reference substance, being configured to concentration with 50% methanol is 20 μ g mL-1's
Reference substance solution, it is spare through 0.45 μm of filtering with microporous membrane to get reference substance solution A；
B, the accurately weighed astragalin of difference, Multiflorin B, afzclin, Multiflorin A reference substance, after mixing
0.8 μ g mL of astragalin concentration is configured to methanol-1, 54 μ g mL of Multiflorin B concentration-1, afzclin concentration
1.5 μg•mL-1, 20 μ g mL of Multiflorin A concentration-1Mixed solution, obtain reference substance solution up to reference substance solution
B, it is spare；
(3) it detects: test solution, reference substance solution A and reference substance solution B injection high performance liquid chromatograph is detected；
(4) quantitative analysis of 5 kinds of flavonoid components in ground centipede medicinal material is carried out: with external standard method with the flavones of known concentration
The chromatographic peak area of class compound compares its respective concentration and carries out regression analysis, obtains standard curve, carries out to test solution
Measurement measures the chromatographic peak area of 5 kinds of flavonoid components in detection test solution, substitutes into standard curve, acquire ground
The content of 5 kinds of flavonoid components in centipede medicinal material.
2. measuring method according to claim 1, the granularity of ground as described in step (1) centipede medicinal powder is 65 mesh.
3. measuring method according to claim 1, methanol water concentration as described in step (1) is 75% methanol.
4. measuring method according to claim 1, as described in step (1) to be heated to reflux the time as 1 h.
5. measuring method according to claim 1, Extracting temperature as described in step (1) is 70 DEG C ± 2 DEG C.
6. measuring method according to claim 1, the high performance liquid chromatograph color of test solution described in step (3)
Spectral condition are as follows:
A, chromatographic column: Waters C18(4.6 nm × 250 mm, 5 μm), mobile phase are as follows: V (methanol): V (water)=35:65 is isocratic
Elution；Flow velocity is 1.0 mLmin -1, column temperature is 25 DEG C, and Detection wavelength is 230 nm, 10 μ L of sample volume；
B, chromatographic column: Waters SunFire C18(4.6 × 250 mm, 5 μm), mobile phase are 0. 2% phosphate aqueous solution
(C)-methanol (D), gradient elution: 0~30 min, C:D=53: 47；30~40 min, 47% D → 66% D；Flow velocity
1.0 mL·min -1, 265 nm of Detection wavelength, 25 DEG C of column temperature, 10 μ L of sample volume.
7. measuring method according to claim 1, the high performance liquid chromatograph of reference substance solution A described in step (3)
Chromatographic condition are as follows:
Chromatographic column: Waters C18(4.6 nm × 250 mm, 5 μm), mobile phase are as follows: V (methanol): V (water)=35:65 is isocratic washes
It is de-；Flow velocity is 1.0 mLmin -1, column temperature is 25 DEG C, and Detection wavelength is 230 nm, 10 μ L of sample volume.
8. measuring method according to claim 1, the high performance liquid chromatograph of reference substance solution B described in step (3)
Chromatographic condition are as follows:
Chromatographic column: Waters SunFire C18(4.6 × 250 mm, 5 μm), mobile phase are 0. 2% phosphate aqueous solution (C)-
Methanol (D), gradient elution: 0~30 min, C:D=53: 47；30~40 min, 47% D → 66% D；Flow velocity 1.0
mL·min -1, 265 nm of Detection wavelength, 25 DEG C of column temperature, 10 μ L of sample volume.
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