CN109374758A - The quantitative finger print atlas and its detection method of phellodendron extract and application - Google Patents
The quantitative finger print atlas and its detection method of phellodendron extract and application Download PDFInfo
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- CN109374758A CN109374758A CN201810942629.6A CN201810942629A CN109374758A CN 109374758 A CN109374758 A CN 109374758A CN 201810942629 A CN201810942629 A CN 201810942629A CN 109374758 A CN109374758 A CN 109374758A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of phellodendron extract quantitative finger print atlas detection methods, this method is established using high performance liquid chromatography, chromatographic condition includes: using octadecylsilane chemically bonded silica as filler, using 0.1 volume % trifluoroacetic acid aqueous solution as mobile phase A, using acetonitrile as Mobile phase B, gradient elution, 0.5~1.5mlmin of flow velocity‑1, 25~35 DEG C of column temperature, 210~360nm of Detection wavelength；Theoretical cam curve is calculated by Berberine hydrochloride peak should be not less than 200,000.In addition, also disclosing the application of finger-print and the finger-print that the detection method obtains.
Description
Technical field
The invention belongs to Pharmaceutical Analysis technical fields, and the construction method of fingerprint image, this method are quantified more particularly to Chinese medicine
It can be used for the qualitative and quantitative study to drug (phellodendron extract) simultaneously.
Background technique
Cortex Phellodendri original name " bark of a cork tree wood ", first recorded in Shennong's Herbal: " bark of a cork tree wood, bitter is cold, main the five internal organs, and heat is tied in stomach, yellow
Subcutaneous ulcer, perianal abscess；Antidiarrheal dysentery, woman's whitish metrorrhagia, yin-yang erosion wound, a Tan Huan, raw mountain valley ".Cortex Phellodendri is bitter in taste, cold.Return kidney, bladder
Through.There is the effect of heat-clearing and damp-drying drug, purging intense heat, except steaming, detoxification sore treatment.For damp-heat dysentery, jaundice, leukorrhagia, heat gonorrhea, tinea pedis , crippling wilt,
Steaming bone and consumptive fever, night sweat, spermatorrhea, sore swollen toxin, eczema itch.Cortex Phellodendri has extensive application in traditional prescription, such as yihuang decoction, clear warp
It dissipates, soup etc. is only drenched in clearing lung-heat.Cortex Phellodendri is also widely used in the modern traditional Chinese patent medicine that the Pharmacopoeia of the People's Republic of China is included simultaneously,
Such as ERMIAO PILL, xiao'er ganyan granules, rotor angle predicting.
Cortex Phellodendri chemical component is based on alkaloid, and one of the main pharmacodynamics ingredient for being considered Cortex Phellodendri, and it is bitter to have seen also Chinese catalpa lemon
Element, phenolic acid class, terpene, lignanoids, sterol and its glycoside etc..Alkaloid compound includes jamaicin (Berberine), bar
Ma Ting (Palmatine), magnoflorine (Magnoflorine), phellodendrine (Phellodendrine), jateorrhizine
(Jatorrhizine), candicine (Candicine), Dauricine (Menisperine).
Quality about Cortex Phellodendri medicinal material controls, and " Chinese Pharmacopoeia " (2015 editions) are by phellodendrine (C20H23NO4), jamaicin
(C20H17NO4) quality control index as the medicinal material.The two ingredients have been carried out containing measurement respectively using the method for efficient liquid phase
It is fixed.Since two indices are to separate measurement, analysis time and analysis cost certainly will be increased.Importantly, in continuous mode
The mobile phase used contains this ion-pairing agent of neopelex.It is found during test, the reagent is not only to change
The retention behavior influence for closing object is bigger and bigger to the damage of chromatographic column.Moreover, for the quality evaluation of Chinese medicine
For, global quality control is crucial.Only the assay of one or two of ingredient can not reflect the real quality of medicinal material.
Currently, Chinese medicine global quality control system is the trend of Chinese medicine development.Finger-print combination multi objective assay
It is the one mode generally approved and be widely used both at home and abroad.
Chinese patent CN103869003A discloses a kind of foundation side of Cortex Phellodendri medicinal material solvent pairs fusion HPLC finger-print
Method and its standard finger-print establish finger-print using high-efficient liquid phase technique, and this method only identifies the chromatographic peak of jamaicin, only
It can be used for the Qualitive test of the medicinal material, the quality good or not of medicinal material can not be evaluated.
Wang Shuhui etc. (" Cortex Phellodendri medicine materical crude slice standard decoction preparation and quality standard research ", " CHINA JOURNAL OF CHINESE MATERIA MEDICA ", volume 43 the
5 phases, in March, 2018, the 873-878 pages) it discloses standard decoction assay and finger-print is established using efficient liquid phase
The method of analysis.Although this be it is a kind of have both qualitative and quantitative detection method, needed in the mobile phase of this method containing
Salt --- ammonium acetate.And only to having pointed out two chromatographic peaks of jamaicin and phellodendrine in finger-print research process.
Therefore, a kind of more preferably flow visualizing is selected, a kind of detection method for capableing of qualitative, quantitative simultaneously is established --- it is fixed
Finger-print is measured, and there are more chromatographic peaks to be pointed out under this condition, for medicinal material total quality detection and whole matter
Amount control is of great significance.
Summary of the invention
Inventor developed a kind of quantitative finger print atlas detection method of phellodendron extract and provide its finger-print.
By the method, while phellodendron extract Berberine and phellodendrine are measured, Cortex Phellodendri medicinal material, medicine materical crude slice, centre can be effectively monitored
The quality of product, preparation guarantees its effective, safety and stabilization in clinical or production applications.
The object of the present invention is to provide a kind of methods of the quantitative finger print atlas of phellodendron extract detection.
The second object of the present invention, which is to provide, to be obtained by the quantitative finger print atlas detection method of above-mentioned phellodendron extract
Finger-print.
The third object of the present invention is to provide the application of above-mentioned finger-print, including can accurately detect phellodendron extract
The content of Berberine and phellodendrine；And Qualitive test can be carried out to phellodendron extract.
In embodiments of the invention, the present invention provides a kind of quantitative finger print atlas detection sides of phellodendron extract
Method, including phellodendron extract is detected using high performance liquid chromatography, wherein chromatographic condition includes:
Using octadecylsilane chemically bonded silica as filler, using 0.1 volume % trifluoroacetic acid aqueous solution as mobile phase A, with
Acetonitrile is Mobile phase B, gradient elution, 0.5~1.5mlmin of flow velocity-1, 25~35 DEG C of column temperature, 210~360nm of Detection wavelength；
Theoretical cam curve is calculated by Berberine hydrochloride peak should be not less than 200,000.
In a preferred embodiment of the invention, the quantitative finger print atlas detection of a kind of phellodendron extract provided by the invention
Method, wherein during gradient elution, the ratio of Mobile phase B changes are as follows: 0~25min, 13 volume % acetonitriles；25~42min,
The 13 volume % acetonitriles of volume %~35；42~50min, the 35 volume % acetonitriles of volume %~95；50~51min, 95 volume %~
13 volume % acetonitriles；51~60min, 13 volume % acetonitriles, i.e. gradient elution program are as follows:
| Time (minute) | Mobile phase A (volume %) | Mobile phase B (volume %) |
| 0~25 | 87 | 13 |
| 25~42 | 87~65 | 13~35 |
| 42~50 | 65~5 | 35~95 |
| 50~51 | 5~87 | 95~13 |
| 51~60 | 87 | 13 |
In a preferred embodiment of the invention, the quantitative finger print atlas detection of a kind of phellodendron extract provided by the invention
Method, wherein the chromatographic condition are as follows: using octadecylsilane chemically bonded silica as filler；With 0.1 volume % trifluoroacetic acid water
Solution is mobile phase A；Using acetonitrile as Mobile phase B；Gradient elution, flow velocity 1.0mlmin-1；30 DEG C of column temperature；Detection wavelength
284nm；Theoretical cam curve is calculated by Berberine hydrochloride peak should be not less than 200,000.
In a preferred embodiment of the invention, the present invention provides a kind of detections of the quantitative finger print atlas of phellodendron extract
Method, wherein described by the chromatographic column of filler of octadecylsilane chemically bonded silica is Agilent SB-C18(specification type
Number be 4.6*250mm, 5 μm).
In a preferred embodiment of the invention, the quantitative finger print atlas detection of a kind of phellodendron extract provided by the invention
Method, further comprising the steps of:
(1) preparation of phellodendron extract: taking Cortex Phellodendri medicine materical crude slice, and 8~12 volumes is added to measure water again, impregnates 0.5~1 hour, heating
It extracts 0.2~1 hour, i.e., one decocts；One, which decocts the rear dregs of a decoction, is added the water that 8~12 volume of Cortex Phellodendri medicine materical crude slice is measured again, extracts 0.2~1 hour,
I.e. two decoct；Merge two to boil medicine liquid, is concentrated under reduced pressure into the concentrate of relative density 1.00~1.15；Concentrate vacuum freeze drying
(freeze temperature is arranged -55 DEG C, and 0.021mbar is arranged in vacuum pressure), obtains freeze-dried powder, freeze-dried powder is further crushed, cross 100 mesh
Sieve, obtains phellodendron extract；
(2) preparation of test solution: taking phellodendron extract 0.1g for step (1), accurately weighed, sets 100ml tool plug cone
In shape bottle, add 60 volume % 25~75ml of methanol aqueous solution, ultrasonic extraction (250w, 40KHz) 10~40 minutes is let cool to room
Temperature is supplied the weight of less loss with 60 volume % methanol aqueous solutions, is shaken up, filtration, take subsequent filtrate to get；
(3) chlorogenic acid reference substance, phellodendrine reference substance, magnoflorine reference substance, jateorrhizine reference substance, jamaicin control are taken
Appropriate product, accurately weighed, respectively plus 60 volume % methanol aqueous solutions are made chromatographic peak and point out and use reference substance solution；
(4) take phellodendrine reference substance, jamaicin reference substance accurately weighed, respectively plus 60 volume % methanol aqueous solutions are made and contain
Reference substance solution is used in measurement surely；
(5) it measures: distinguishing accurate aspiration step (3) chromatographic peak and point out with reference substance solution, step (4) assay use pair
According to product solution and each 2~10 μ l of step (2) test solution, high performance liquid chromatograph, the chromatography in record 60 minutes are injected
Figure calculates the percentage composition (respectively in terms of hydrochloric acid phellodendrine, Berberine hydrochloride) of phellodendrine and jamaicin in phellodendron extract, uses
" similarity evaluation " 2012.130723 editions handles to get phellodendron extract map
Finger-print.
In more preferred of the invention, the present invention provides a kind of inspections of the quantitative finger print atlas of phellodendron extract
Survey method, comprising the following steps:
(1) acquisition modes of phellodendron extract are as follows: Cortex Phellodendri medicine materical crude slice is taken, 10 volumes is added to measure water again, is impregnated 0.5 hour, heating
It extracts 0.5 hour, i.e., one decocts；One, which decocts the rear dregs of a decoction, is added the water that 8 volume of Cortex Phellodendri medicine materical crude slice is measured again, extracts 20 minutes, i.e., two decoct；Merge
Two boil medicine liquid, are concentrated under reduced pressure into the concentrate of relative density 1.1 or so；Concentrate vacuum freeze drying, freeze temperature setting -55
DEG C, 0.021mbar is arranged in vacuum pressure, obtains freeze-dried powder, crushes, sieves with 100 mesh sieve, obtain phellodendron extract；
(2) preparation of test solution: taking phellodendron extract fine powder 0.1g for step (1), accurately weighed, sets 100ml tool
It fills in conical flask, precision plus 60 volume % methanol aqueous solution 50ml, ultrasonic extraction (250w, 40KHz) 20 minutes are let cool to room
Temperature is supplied the weight of less loss with 60 volume % methanol aqueous solutions, is shaken up, filtration, take subsequent filtrate to get；
(3) chromatographic peak points out the preparation with reference substance solution: taking chlorogenic acid reference substance, phellodendrine reference substance, magnoflorine
Reference substance, jateorrhizine reference substance, jamaicin reference substance are appropriate, accurately weighed, add 60 volume % methanol aqueous solutions that every 1ml is made and contain
The mixed reference substance solution of 20 μ g of chlorogenic acid, 40 μ g of phellodendrine, 15 μ g of magnoflorine, 5 μ g of jateorrhizine, jamaicin 0.3mg, i.e.,
?；
(4) preparation of assay reference substance solution: taking hydrochloric acid phellodendrine, Berberine hydrochloride reference substance appropriate, accurate
It is weighed, add 60 volume % methanol aqueous solutions that the mixing pair of hydrochloric phellodendrine 40 μ g, Berberine hydrochloride 0.3mg in every 1ml is made
According to product solution to get；
(5) it measures: distinguishing accurate aspiration step (3) chromatographic peak and point out with reference substance solution, step (4) assay use pair
According to product solution and each 10 μ l of step (2) test solution, high performance liquid chromatograph is injected, records the chromatogram in 60 minutes,
Using external standard peak area method, while the content of phellodendrine and jamaicin is measured in phellodendron extract (respectively with hydrochloric acid phellodendrine, salt
Sour jamaicin meter), map is handled with " similarity evaluation " 2012.130723 editions, i.e.,
Obtain the finger-print of phellodendron extract；
Here, the chromatographic condition of the high performance liquid chromatography measurement are as follows: using octadecylsilane chemically bonded silica as filler；
Using 0.1 volume % trifluoroacetic acid aqueous solution as mobile phase A, using acetonitrile as Mobile phase B；Gradient elution, flow velocity 1.0mlmin-1；
30 DEG C of column temperature；Detection wavelength 284nm；Theoretical cam curve is calculated by Berberine hydrochloride peak should be not less than 200,000.
On the other hand, the present invention provides the Huangs that the quantitative finger print atlas detection method by above-mentioned phellodendron extract obtains
Cypress extract finger-print has 14 shared peaks in the map；Wherein, the retention time at each shared peak is respectively as follows: shared peak
1:4.603min；Shared peak 2:4.976min；Shared peak 3:7.493min；Shared peak 4:8.195min；Shared peak 5:
8.574min；Shared peak 6:11.441min；Shared peak 7:17.175min；Shared peak 8:19.200min；Shared peak 9:
24.882min；Shared peak 10:26.171min；Shared peak 11:29.064min；Shared peak 12:40.479min；Shared peak 13:
42.896min；Shared peak 14:46.245min.Chlorogenic acid corresponds to No. 3 shared peaks, phellodendrine corresponding No. 8 shared peaks, magnoflorines
The corresponding No. 13 shared peaks of corresponding No. 10 shared peaks, jateorrhizine, the corresponding No. 14 shared peaks of jamaicin.
The third aspect, the present invention provides above-mentioned phellodendron extract finger-print detection phellodendron extract Berberine and
The application of Cortex Phellodendri alkali content and above-mentioned phellodendron extract finger-print carry out the application of Qualitive test to phellodendron extract.?
That is using the finger-print assay, the height of content can be carried out to two effective components in phellodendron extract simultaneously
Just reflect the superiority and inferiority of extract quality；It is true that the liquid chromatogram that assay process obtains simultaneously can also be used in phellodendron extract
Pseudo- identification, by compare the spectrogram of extract with compare map similarity just and can be seen that the true and false and adulterated situation of extract.
Pass through the assay carried out to the effective component in 18 batches of phellodendron extracts, phellodendrine (in terms of hydrochloric acid phellodendrine)
Average content is 16.22mgg-1, jamaicin (in terms of Berberine hydrochloride) average content is 108.45mgg-1。
The beneficial effects of the present invention are:
The map that detection method measures can more comprehensively reflect the chemical component in phellodendron extract, and each chromatography
Peak separation preferably, baseline is steady, and peak type is good, repeatability preferably, this method have good linear relationship, precision, stability,
The rate of recovery can accurately detect the component content of phellodendron extract.The present invention provides and passes through according to 9 18 batches, place of production samples
The phellodendron extract quantitative finger print atlas that phellodendron extract quantitative finger print atlas detection method as described above obtains.
In embodiments of the invention, it is related to " % ", unless otherwise specified, all refers to percent by volume.
Detailed description of the invention
Attached drawing is used to provide to further understand technical solution of the present invention, and constitutes part of specification, with this
The embodiment of application technical solution for explaining the present invention together, does not constitute the limitation to technical solution of the present invention.
Fig. 1 is chromatogram of the flow visualizing used herein compared with other flow visualizings；- 0.1 body of A acetonitrile
Product % aqueous formic acid (8mM ammonium acetate)；- 1 volume % aqueous acetic acid of B acetonitrile；C acetonitrile-water；- 0.05 volume % phosphorus of D acetonitrile
Aqueous acid；- 1 volume % aqueous formic acid of E acetonitrile；- 0.1 volume % trifluoroacetic acid aqueous solution of F acetonitrile；1- phellodendrine；2- is small
Bark of a cork tree alkali.
Fig. 2 is 18 crowdes of phellodendron extract finger-print stacking charts that the present invention measures.
Fig. 3 is the control map for measuring phellodendron extract finger-print through the invention.
Fig. 4 is the phellodendron extract typical case's test solution finger-print measured through the invention, when abscissa is in figure
Between (min), ordinate be absorbance (AU).
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Embodiment 1: Cortex Phellodendri medicinal material acquires information
1. Cortex Phellodendri of table acquires information table
Embodiment 2: phellodendron extract finger-print
The construction method of the quantitative finger print atlas of phellodendron extract surveys phellodendron extract using liquid chromatography
Fixed, the specific method is as follows:
(1) chromatographic peak points out the preparation with reference substance solution
Take chlorogenic acid reference substance, phellodendrine reference substance, magnoflorine reference substance, jateorrhizine reference substance, jamaicin reference substance
In right amount, accurately weighed, add 60 volume % methanol aqueous solutions that every 1ml is made containing 20 μ g of chlorogenic acid, 40 μ g of phellodendrine, magnoflorine 15
μ g, 5 μ g of jateorrhizine, jamaicin 0.3mg mixed reference substance solution to get.
(2) preparation of assay reference substance solution
Take hydrochloric acid phellodendrine, Berberine hydrochloride reference substance appropriate, it is accurately weighed, add 60 volume % methanol aqueous solutions to be made often
Hydrochloric 40 μ g of phellodendrine in 1ml, Berberine hydrochloride 0.3mg mixed reference substance solution to get.
(3) preparation of test solution
Phellodendron extract fine powder 0.1g is taken, it is accurately weighed, it sets in 100ml stuffed conical flask, precision plus 60 volume % methanol
Aqueous solution 50ml, ultrasonic extraction (250w, 40KHz) 20 minutes, lets cool to room temperature, supplies less loss with 60 volume % methanol aqueous solutions
Weight, shake up, filter, take subsequent filtrate to get；
(4) chromatographic condition and system suitability
Chromatographic column: Agilent SB-C18(specifications and models 4.6*250mm, 5 μm)
Mobile phase: A pump: 0.1 volume % trifluoroacetic acid aqueous solution；B pump: acetonitrile；
Gradient elution program:
Detection wavelength: 284nm；
Flow velocity: 1.0mlmin-1；
Column temperature: 30 DEG C
Theoretical cam curve is not less than 200,000 based on Berberine hydrochloride peak.
(1) it measures: it is accurate respectively to draw reference substance solution and each 10 μ l of test solution, high performance liquid chromatograph is injected,
Chromatogram in record 60 minutes.Two indices component content is calculated, with " chromatographic fingerprints of Chinese materia medica similarity evaluation system
System " 2012.130723 editions finger-prints map handled to get phellodendron extract.See Fig. 2 and Fig. 3.
Liquid chromatograph is 1260 Infinity Series high performance liquid chromatograph of Agilent.
There are 14 shared peaks in finger-print, detected simultaneously by reference substance solution and test solution, identifies 5 colors
Spectral peak.Wherein No. 3 peaks are chlorogenic acid, No. 8 peaks are phellodendrine, No. 10 peaks be magnoflorine, No. 13 peaks be jateorrhizine, No. 14 peaks
For jamaicin.See Fig. 4.
Pass through the assay carried out to the effective component in 18 batches of phellodendron extracts, phellodendrine (in terms of hydrochloric acid phellodendrine)
Average content is 16.22mgg-1, jamaicin (in terms of Berberine hydrochloride) average content is 108.45mgg-1。
Preparation method of test article is investigated
(1) investigation of Extraction solvent
Respectively investigate water, 20 volume % methanol aqueous solutions, 40 volume % methanol aqueous solutions, 60 volume % methanol aqueous solutions,
80 volume % methanol aqueous solutions, pure methanol, hydrochloric acid: methanol (1:100, v/v), acetic acid: methanol (1:100, v/v), -70 body of hydrochloric acid
Product % ethanol water (1:100, v/v).40 volume % methanol aqueous solutions, 60 volume % methanol aqueous solutions extract effect as the result is shown
Fruit is suitable, and in terms of the difficulty or ease that miillpore filter is crossed in test sample treatment process, 60 volume % methanol aqueous solutions are more preferably.
(2) investigation of different extracting modes
Three kinds of refluxing extraction, ultrasonic extraction, mechanical shaking extraction extracting modes are compared, refluxing extraction, ultrasound mention as the result is shown
No significant difference is taken, extracting mode of the ultrasonic extraction as phellodendron extract is selected.
(3) investigation of different solvents volume
The Extraction solvent of tri- kinds of different volumes of 25ml, 50ml, 75ml is investigated respectively, as the result is shown different solvents body
Long-pending extraction efficiency is without significant difference, it is contemplated that the peak area of phellodendrine, jamaicin, therefore select volume for 50ml.
(4) investigation of different extraction times
Investigated the extraction time of 10min, 20min, 40min as phellodendron extract respectively, as a result extraction time be
20min extraction efficiency is relatively high, selects 20min for the ultrasonic extraction time of phellodendron extract.
The investigation of phellodendron extract chromatographic condition
(1) investigation of variety classes acid flow visualizing
It compared -0.1 volume % aqueous formic acid (ammonium acetate containing 8mM) (system A) of acetonitrile, -1 volume % acetic acid water of acetonitrile
Solution (system B), acetonitrile-water (system C), -0.05 volume % phosphate aqueous solution (system D) of acetonitrile, -1 volume % formic acid of acetonitrile
Aqueous solution (system E), -0.1 volume % trifluoroacetic acid aqueous solution (system F) of acetonitrile this 6 kinds of flow visualizings, are shown in Fig. 1.It was found that not
The peak sequence of phellodendrine chromatographic peak is influenced with flow visualizing very big.It is from Fig. 1 it can be found that attached in No. 1 peak (phellodendrine)
Closely there is more apparent unknown peak a --- peak 3, only when using acetonitrile-trifluoroacetic acid system, phellodendrine can be at peak 3
Appearance below, and use phellodendrine appearance before peak 3 when other flow visualizings.It can be seen that having very in phellodendron extract
More small peaks all concentrate on before peak 3, if the phellodendrine also appearance before peak 3, will necessarily wrap up, therefore make with impurity small peak
When with B, C, D, E system, phellodendrine can not achieve baseline separation.By the system suitability parameter for comparing the two systems of A, F
It is not difficult to find that the separating degree of phellodendrine and previous impurity small peak is 3.1, and A system is 1.5 when using F system, illustrate to use F
When system, the separation better off of phellodendrine；Moreover, F system also improves the separation situation of jamaicin, so that jamaicin
With previous impurity small peak baseline separation, separating degree reaches 2.1, meanwhile, the case where jamaicin trails, is also improved, hangover because
Son is 1.57.It can be seen that the separating effect of -0.1 volume % trifluoroacetic acid aqueous solution (system F) of acetonitrile is more preferable.
Table 2.A/F architecture system adaptability-mobile phase selection
(2) investigation of different acid concentrations
It compared 0.05 volume % trifluoroacetic acid, 0.1 volume % trifluoroacetic acid, 0.15 volume % trifluoroacetic acid respectively, tie
0.05 volume % trifluoroacetic acid of fruit, 0.15 volume % trifluoroacetic acid separating effect are preferable, and in view of acidity is excessively high to chromatographic column
It has damage, 0.1 volume % trifluoroacetic acid aqueous solution of selection is mobile phase.
(3) investigation of different chromatographic columns
It compared Agilent SB C respectively18(4.6*250mm, 5 μm), Kromasil C18(4.6*250mm, 5 μm) two
The C18 chromatographic column of root different brands finds being applicable in for this method.In view of mobile phase is in acidity, therefore selection is suitble in low pH
The Agilent SB C used under the conditions of value18When (4.6*250mm, 5 μm) is analyzed as phellodendron extract quantitative finger print atlas
Chromatographic column.
(4) investigation of different column temperatures
It compared 25 DEG C, 30 DEG C, 35 DEG C of three kinds of column temperatures, as a result column temperature increases, and separating degree becomes smaller.Comprehensively consider separating degree with
Retention time selects column temperature for 30 DEG C.
(5) investigation of different wave length
The a length of 284nm of phellodendrine maximum absorption wave, a length of 264nm of jamaicin maximum absorption wave.This method is intended to survey simultaneously
Determine the content of jamaicin in Cortex Phellodendri standard decoction freeze-dried powder, phellodendrine, the finger-print chromatographic peak information under 284nm compares
It is more, comprehensively consider the Detection wavelength for selecting 284nm as the analysis of phellodendron extract quantitative finger print atlas when.
Methodological study
(1) repeatability is investigated
Taking lot number is the phellodendron extract 0.1g of CPCSD-14, prepares 6 parts of test solutions according to preparation method of test article.
According to above-mentioned chromatographic condition sample introduction, chromatogram is recorded.Calculate the percentage composition (difference of phellodendrine and jamaicin in phellodendron extract
In terms of hydrochloric acid phellodendrine, Berberine hydrochloride), and similarity-rough set is carried out to 6 maps.The result shows that test solution middle finger
Index composition content is almost the same (RSD < 3%), and the similarity between map is 1.000, shows that this method repeatability is good, is shown in Table
3、4。
3. phellodendron extract assay index components repeatability of table investigates result (mass percentage)
4. phellodendron extract finger-print repeatability of table investigates result
(2) linear relationship, range
Precision weighs phellodendrine and jamaicin reference substance is appropriate, adds 60 volume % methanol aqueous solutions that every 1ml is made containing Cortex Phellodendri
The mixing mother liquor of alkali 200 μ g and jamaicin 0.7mg.Precision measure 4,3,2,1ml reference substance mother liquor be placed in 5ml measuring bottle, 1ml pairs
It is placed in 10ml measuring bottle according to product mother liquor, 0.5ml reference substance mother liquor is placed in 20ml measuring bottle, and 60 volume % methanol aqueous solutions is added to dilute
To scale, shake up to get.With reference substance concentration (μ g.ml-1) it is abscissa, peak area is ordinate, is carried out with least square method
Linear regression.Each reference substance range of linearity, regression equation and related coefficient are shown in Table 5.
Each reference substance range of linearity of table 5., regression equation and related coefficient
(3) accuracy
Taking lot number is the phellodendron extract 0.05g of CPCSD-14, accurately weighed, is set in 100ml volumetric flask, it is accurate respectively plus
Enter mixed reference substance solution (hydrochloric acid phellodendrine 0.22mg.ml-1, Berberine hydrochloride 1.52mg.ml-1) 2,4,6ml, 60 bodies are added
Product % methanol aqueous solution constant volume, close plug are ultrasonically treated 20 minutes, let cool, the weight of less loss is supplied with 60 volume % methanol aqueous solutions
Amount, shake up, miillpore filter filtration, take subsequent filtrate to get.Measurement calculates the rate of recovery.It the results are shown in Table 6, phellodendrine, jamaicin return
The numerical value of yield meets regulation in 94%~97%, 96%~99% (RSD < 3%).Show this method energy Accurate Determining sample
Middle phellodendrine, content of berberine.
6. two indices component content accuracy of measurement of table investigates result
(4) Intermediate precision
Under another date, it is the phellodendron extract 0.05g of CPCSD-14 that another one analysis personnel, which take lot number, prepares six parts
Test solution, according to above-mentioned chromatographic condition on another high performance liquid chromatograph sample introduction, record chromatogram.Cortex Phellodendri is calculated to extract
The percentage composition (respectively in terms of hydrochloric acid phellodendrine, Berberine hydrochloride) of phellodendrine and jamaicin in object merges repeated data, meter
It calculates, and Supplements similarity-rough set is carried out to 12 maps.The result shows that the RSD of Cortex Phellodendri alkali content is 3.22% (< 4%),
The RSD of content of berberine is 0.45% (< 3%), and the similarity between map is 1.000, shows that this method precision is good, sees
Table 7,8.
7. phellodendron extract assay index components precision of table investigates result (mass percentage)
8. phellodendron extract finger-print precision of table investigates result
(5) study on the stability
Taking lot number is the phellodendron extract 0.05g of CPCSD-14, prepares test solution according to preparation method of test article, point
0h, 2h, 4h, 8h, 12h, for 24 hours sample introduction not after sample preparation.Chromatogram is recorded, phellodendrine and jamaicin in phellodendron extract are calculated
Percentage composition (respectively in terms of hydrochloric acid phellodendrine, Berberine hydrochloride), and similarity-rough set is carried out to the full peak of 7 maps.As a result
Show that two indices component content is almost the same (RSD < 3%) in test solution, the similarity between map is 1.000, table
Bright sample stability is good, is shown in Table 9,10.
9. phellodendron extract assay index components study on the stability result (percentage composition) of table
10. phellodendron extract finger-print study on the stability result of table
Similarity-rough set
9 18 batches, place of production phellodendron extracts, prepare test solution by the preparation method of above-mentioned test solution, measurement.
Chromatogram is recorded, the percentage composition of phellodendrine and jamaicin is (respectively with hydrochloric acid phellodendrine, hydrochloric acid barberry in calculating phellodendron extract
Alkali meter).The HPLC map of 18 batches of phellodendron extracts successively imports " chromatographic fingerprints of Chinese materia medica similarity evaluation system with AIA format
System ", similarity comparison is carried out with the reference fingerprint of generation, the results are shown in Table 11,12.
11. phellodendron extract assay result (mg.g of table-1)
12.18 batches of phellodendron extract similarity evaluation results of table
It is above-mentioned statistics indicate that, the similarity of 18 batches of phellodendron extracts and reference fingerprint is 0.95 or more.
The map that detection method measures can more comprehensively reflect the chemical component in phellodendron extract, and each chromatography
Peak separation preferably, baseline is steady, and peak type is good, repeatability preferably, this method have good linear relationship, precision, stability,
The rate of recovery can accurately detect the component content of phellodendron extract.By the method can effectively monitor Cortex Phellodendri medicinal material, medicine materical crude slice,
The quality of intermediate product, preparation guarantees its effective, safety and stabilization in clinical or production applications.
The above is only exemplary embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member for, without departing from the principle of the present invention, improvement and optimization can also be made according to the prior art, these improve and
Optimization also belongs to protection scope of the present invention.
Claims (10)
1. a kind of quantitative finger print atlas detection method of phellodendron extract, including using high performance liquid chromatography to phellodendron extract
It is detected, wherein chromatographic condition includes:
Chromatographic column: using octadecylsilane chemically bonded silica as filler；Using 0.1 volume % trifluoroacetic acid aqueous solution as mobile phase A,
Using acetonitrile as Mobile phase B, gradient elution, 0.5~1.5mlmin of flow velocity-1, 25~35 DEG C of column temperature, Detection wavelength 210~
360nm；Theoretical cam curve is calculated by Berberine hydrochloride peak should be not less than 200,000.
2. detection method as described in claim 1, wherein during gradient elution, the ratio of Mobile phase B changes are as follows: 0~
25min, 13 volume % acetonitriles；25~42min, the 13 volume % acetonitriles of volume %~35；42~50min, 35 bodies of volume %~95
Product % acetonitrile；50~51min, the 95 volume % acetonitriles of volume %~13；51~60min, 13 volume % acetonitriles.
3. detection method as described in claim 1, wherein the chromatographic condition are as follows: be with octadecylsilane chemically bonded silica
Filler；Using 0.1 volume % trifluoroacetic acid aqueous solution as mobile phase A, using acetonitrile as Mobile phase B；Gradient elution, flow velocity
1.0ml·min-1；30 DEG C of column temperature；Detection wavelength 284nm；Theoretical cam curve is calculated by Berberine hydrochloride peak should be not less than 200,
000。
4. detection method as described in claim 1, wherein be by the chromatographic column of filler of octadecylsilane chemically bonded silica
Agilent SB C18, specifications and models are as follows: 4.6*250mm, 5 μm.
5. further comprising the steps of such as detection method of any of claims 1-4:
(1) preparation of phellodendron extract: taking Cortex Phellodendri medicine materical crude slice, and 8~12 volumes is added to measure water again, impregnates 0.5~1 hour, heating extraction
0.2~1 hour, i.e., one decocts；One decocts the water that 8~12 times of Cortex Phellodendri medicine materical crude slice amounts are added in the rear dregs of a decoction, extracts 0.2~1 hour, i.e., two decoct；
Merge two to boil medicine liquid, is concentrated under reduced pressure into the concentrate of relative density 1.00~1.15；Concentrate vacuum freeze drying must be lyophilized
Freeze-dried powder is further crushed, sieves with 100 mesh sieve, obtain phellodendron extract by powder；
(2) preparation of test solution: taking phellodendron extract 0.1g, accurately weighed, sets in 100ml stuffed conical flask, adds 60 bodies
Product 25~75ml of % methanol aqueous solution, ultrasonic extraction 10~40 minutes, lets cool to room temperature, is supplied with 60 volume % methanol aqueous solutions
The weight of less loss, shakes up, filtration, take subsequent filtrate to get；
(3) chromatographic peak points out the preparation with reference substance solution: taking chlorogenic acid reference substance, phellodendrine reference substance, magnoflorine control
Product, jateorrhizine reference substance, jamaicin reference substance are appropriate, accurately weighed, and respectively plus chromatographic peak is made in 60 volume % methanol aqueous solutions
It points out and uses reference substance solution；
(4) preparation of assay reference substance solution: taking phellodendrine reference substance, jamaicin reference substance accurately weighed, respectively plus
Assay reference substance solution is made in 60 volume % methanol aqueous solutions；
(5) measure: accurate aspiration step (3) chromatographic peak is pointed out with reference substance solution, step (4) assay reference substance respectively
Solution and each 2~10 μ l of step (2) test solution inject high performance liquid chromatograph, the chromatogram in record 60 minutes；
The percentage composition (respectively in terms of hydrochloric acid phellodendrine, Berberine hydrochloride) for calculating phellodendrine and jamaicin in phellodendron extract, with " in
Medicine chromatographic fingerprinting similarity evaluation system " 2012.130723 editions map handled quantified to get phellodendron extract
Finger-print.
6. detection method as claimed in claim 5, comprising the following steps:
(1) acquisition modes of phellodendron extract are as follows: take Cortex Phellodendri medicine materical crude slice, 10 volumes is added to measure water again, impregnate 0.5 hour, heating extraction
0.5 hour, i.e., one decocts；One, which decocts the rear dregs of a decoction, is added the water that 8 volume of Cortex Phellodendri medicine materical crude slice is measured again, extracts 20 minutes, i.e., two decoct；Merge two to decoct
Medical fluid is concentrated under reduced pressure into the concentrate of relative density 1.1；Concentrate vacuum freeze drying, freeze temperature are arranged -55 DEG C, vacuum
0.021mbar is arranged in pressure, obtains freeze-dried powder, freeze-dried powder is further crushed, sieves with 100 mesh sieve, obtains phellodendron extract；
(2) preparation of test solution: taking phellodendron extract 0.1g, accurately weighed, sets in 100ml stuffed conical flask, adds 60 bodies
Product % methanol aqueous solution 50ml, ultrasonic extraction (250w, 40KHz) 20 minutes is let cool to room temperature, with 60 volume % methanol aqueous solutions
The weight for supplying less loss, shakes up, filtration, take subsequent filtrate to get；
(3) chromatographic peak points out the preparation with reference substance solution: taking chlorogenic acid reference substance, phellodendrine reference substance, magnoflorine control
Product, jateorrhizine reference substance, jamaicin reference substance are appropriate, accurately weighed, add 60 volume % methanol aqueous solutions that every 1ml is made containing green original
20 μ g of acid, 40 μ g of phellodendrine, 15 μ g of magnoflorine, 5 μ g of jateorrhizine, jamaicin 0.3mg mixed reference substance solution to get；
(4) preparation of assay reference substance solution: taking hydrochloric acid phellodendrine, Berberine hydrochloride reference substance appropriate, accurately weighed,
Add 60 volume % methanol aqueous solutions that hydrochloric 40 μ g of phellodendrine, the mixing reference substance of Berberine hydrochloride 0.3mg in every 1ml is made molten
Liquid to get；
(5) measure: accurate aspiration step (3) chromatographic peak is pointed out with reference substance solution, step (4) assay reference substance respectively
Solution and each 2~10 μ l of step (2) test solution inject high performance liquid chromatograph, the chromatogram in record 60 minutes；
Using external standard peak area method, while the content of phellodendrine and jamaicin is measured in phellodendron extract (respectively with hydrochloric acid phellodendrine, salt
Sour jamaicin meter), map is handled with " similarity evaluation " 2012.130723 editions, i.e.,
Obtain the quantitative finger print atlas of phellodendron extract.
7. the Cortex Phellodendri as the quantitative finger print atlas detection method of phellodendron extract of any of claims 1-6 obtains mentions
Object quantitative finger print atlas is taken, there are 14 shared peaks in the map.
8. finger-print as claimed in claim 7, wherein the retention time at each shared peak is respectively as follows: shared peak 1:
4.603min；Shared peak 2:4.976min；Shared peak 3:7.493min；Shared peak 4:8.195min；Shared peak 5:8.574min；
Shared peak 6:11.441min；Shared peak 7:17.175min；Shared peak 8:19.200min；Shared peak 9:24.882min；It is shared
Peak 10:26.171min；Shared peak 11:29.064min；Shared peak 12:40.479min；Shared peak 13:42.896min；It is shared
Peak 14:46.245min.
9. finger-print as claimed in claim 7 or 8 is in the application of detection phellodendron extract Berberine and Cortex Phellodendri alkali content.
10. finger-print as claimed in claim 7 or 8 is carrying out the application in Qualitive test to phellodendron extract.
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