CN109828064A - A kind of HPLC fingerprint image method for building up of Heiguteng exract effective part group - Google Patents
A kind of HPLC fingerprint image method for building up of Heiguteng exract effective part group Download PDFInfo
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- CN109828064A CN109828064A CN201910016854.1A CN201910016854A CN109828064A CN 109828064 A CN109828064 A CN 109828064A CN 201910016854 A CN201910016854 A CN 201910016854A CN 109828064 A CN109828064 A CN 109828064A
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- 238000000034 method Methods 0.000 title claims abstract description 39
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 33
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 66
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 39
- PZIRUHCJZBGLDY-UHFFFAOYSA-N Caffeoylquinic acid Natural products CC(CCC(=O)C(C)C1C(=O)CC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C)C(=O)O PZIRUHCJZBGLDY-UHFFFAOYSA-N 0.000 claims description 29
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
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- 238000010828 elution Methods 0.000 claims description 19
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- 235000001368 chlorogenic acid Nutrition 0.000 claims description 18
- CWVRJTMFETXNAD-FWCWNIRPSA-N 3-O-Caffeoylquinic acid Natural products O[C@H]1[C@@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-FWCWNIRPSA-N 0.000 claims description 16
- CWVRJTMFETXNAD-KLZCAUPSSA-N Neochlorogenin-saeure Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O CWVRJTMFETXNAD-KLZCAUPSSA-N 0.000 claims description 16
- CWVRJTMFETXNAD-JUHZACGLSA-N chlorogenic acid Chemical compound O[C@@H]1[C@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-JUHZACGLSA-N 0.000 claims description 16
- 229940074393 chlorogenic acid Drugs 0.000 claims description 16
- FFQSDFBBSXGVKF-KHSQJDLVSA-N chlorogenic acid Natural products O[C@@H]1C[C@](O)(C[C@@H](CC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O FFQSDFBBSXGVKF-KHSQJDLVSA-N 0.000 claims description 16
- BMRSEYFENKXDIS-KLZCAUPSSA-N cis-3-O-p-coumaroylquinic acid Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)cc2)[C@@H]1O)C(=O)O BMRSEYFENKXDIS-KLZCAUPSSA-N 0.000 claims description 16
- 239000013558 reference substance Substances 0.000 claims description 16
- 238000012360 testing method Methods 0.000 claims description 16
- 239000012488 sample solution Substances 0.000 claims description 15
- 239000012153 distilled water Substances 0.000 claims description 14
- UFCLZKMFXSILNL-BBLPPJRLSA-N (-) 4,5-dicaffeoylquinic acid Natural products OC=1C=C(C=CC=1O)C=CC(=O)O[C@@H]1C[C@@](C[C@H]([C@H]1OC(C=CC1=CC(=C(C=C1)O)O)=O)O)(C(=O)O)O UFCLZKMFXSILNL-BBLPPJRLSA-N 0.000 claims description 13
- UFCLZKMFXSILNL-BKUKFAEQSA-N 3,4-di-O-caffeoylquinic acid Natural products O[C@H]1C[C@](O)(C[C@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1OC(=O)C=Cc3ccc(O)c(O)c3)C(=O)O UFCLZKMFXSILNL-BKUKFAEQSA-N 0.000 claims description 13
- UFCLZKMFXSILNL-PSEXTPKNSA-N Isochlorogenic acid b Chemical compound O([C@@H]1C[C@@](O)(C[C@H]([C@H]1OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)O)C(O)=O)C(=O)\C=C\C1=CC=C(O)C(O)=C1 UFCLZKMFXSILNL-PSEXTPKNSA-N 0.000 claims description 13
- UFCLZKMFXSILNL-UHFFFAOYSA-N NSC 649410 Natural products C=1C=C(O)C(O)=CC=1C=CC(=O)OC1C(O)CC(O)(C(O)=O)CC1OC(=O)C=CC1=CC=C(O)C(O)=C1 UFCLZKMFXSILNL-UHFFFAOYSA-N 0.000 claims description 13
- 239000000706 filtrate Substances 0.000 claims description 13
- GWTUHAXUUFROTF-UHFFFAOYSA-N pseudochlorogenic acid Natural products C1C(O)C(O)C(O)CC1(C(O)=O)OC(=O)C=CC1=CC=C(O)C(O)=C1 GWTUHAXUUFROTF-UHFFFAOYSA-N 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- 239000012085 test solution Substances 0.000 claims description 13
- CWVRJTMFETXNAD-NXLLHMKUSA-N trans-5-O-caffeoyl-D-quinic acid Chemical compound O[C@H]1[C@H](O)C[C@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-NXLLHMKUSA-N 0.000 claims description 13
- GYFFKZTYYAFCTR-JUHZACGLSA-N 4-O-trans-caffeoylquinic acid Chemical compound O[C@@H]1C[C@](O)(C(O)=O)C[C@@H](O)[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 GYFFKZTYYAFCTR-JUHZACGLSA-N 0.000 claims description 11
- GYFFKZTYYAFCTR-UHFFFAOYSA-N 5-O-(6'-O-galloyl)-beta-D-glucopyranosylgentisic acid Natural products OC1CC(O)(C(O)=O)CC(O)C1OC(=O)C=CC1=CC=C(O)C(O)=C1 GYFFKZTYYAFCTR-UHFFFAOYSA-N 0.000 claims description 11
- 229910019142 PO4 Inorganic materials 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 11
- GYFFKZTYYAFCTR-LMRQPLJMSA-N cryptochlorogenic acid Natural products O[C@H]1C[C@@](O)(C[C@H](O)[C@H]1OC(=O)C=Cc2ccc(O)c(O)c2)C(=O)O GYFFKZTYYAFCTR-LMRQPLJMSA-N 0.000 claims description 11
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 11
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- Measurement Of The Respiration, Hearing Ability, Form, And Blood Characteristics Of Living Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a kind of method for building up of the HPLC fingerprint image of Heiguteng exract effective part group.This method has easy, stable, precision height, high repeatability and other advantages；It more can also comprehensively reflect the type and quantity of contained chemical component in Heiguteng exract effective part group with this method, and then whole description and evaluation are carried out to its quality.And the Heiguteng exract active component group fingerprint atlas chemical component that is detected with this method is relatively more, each characteristic peak height ratio is moderate, baseline is more steady, and separating degree, peak shape are preferable, and column effect is higher.
Description
Technical field
The present invention relates to a kind of method for building up of the HPLC fingerprint image of Heiguteng exract effective part group, belong to the neck of drug technology
Domain.
Background technique
Heiguteng exract (Periploca forrestii Schltr.) belongs to Asclepiadaceae (Asclepiadaceae) Periploca
(Periploca) plant is recorded in 2003 editions " Guizhou Province's Chinese medicine, Ethnic crude drugs quality standards ", has dispelling wind and eliminating dampness, promoting blood circulation
The function that dredging collateral, the numbness that disappears relieve pain, can relaxing tendons and activating collaterals, dispelling wind and eliminating dampness；Cure mainly rheumatic arthritis, traumatic injury, stomachache, digestion not
Good, amenorrhoea, dysentery etc..Heiguteng exract is one of common seedling medicine, is that Heiguteng exract chases after wind collaterals-activating capsule, tendon-extending bone-penetrating liquid, black
Bone rattan stretches the important composition flavour of a drug of muscle penetrating spray.
Traditional Chinese medicine fingerprint is a kind of synthesis, quantifiable identification of means, it is built upon chemical composition of Chinese materia medica system
On the basis of research, it is mainly used for evaluating authenticity, Optimality and the stability of Chinese medicine and Chinese materia medica preparation semi-manufactured goods quality.
Chinese medicine and its preparation are multi-component complex system, therefore evaluate its quality and can should be provided to enrich and be reflected using adaptable therewith
The detection method of other information, establishing traditional Chinese medicine fingerprint more will comprehensively reflect contained chemical component in Chinese medicine and its preparation
Type and quantity, and then whole description and evaluation are carried out to drug quality.On this basis, it is learned if further carrying out spectrum effect
Research, can be such that traditional Chinese medicine quality really combines with its drug effect, help to illustrate Mechanism of TCM.Currently, there is no Heiguteng exract
The report of effective part group HPLC finger-print research work.
Summary of the invention
Present invention aims at provide a kind of method for building up of the HPLC fingerprint image of Heiguteng exract effective part group.This method
With easy, stable, precision is high, high repeatability and other advantages；More can also comprehensively it be reflected with this method contained in Heiguteng exract
The type and quantity of chemical component, and then whole description and evaluation are carried out to its quality.And the Heiguteng exract detected with this method
Finger-print chemical component is relatively more, each characteristic peak height ratio is moderate, and baseline is more steady, and separating degree, peak shape are preferable, column effect
It is higher.
In order to solve the above technical problems, the present invention adopts the following technical scheme that realization: a kind of Heiguteng exract effective part group
HPLC fingerprint image method for building up, comprising the following steps:
(1) preparation of mixed reference substance solution: taking neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid and 4,5-Dicaffeoylquinic acid standard items,
It is neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid and 4,5-Dicaffeoylquinic acid concentration with methanol constant volume is 0.005-0.015gmL-1Mixing
Reference substance solution；
(2) preparation of test solution: accurately weighed Heiguteng exract coarse powder 100.0g is extracted with alcohol reflux, is filtered, dense
Contracting, with distilled water constant volume 1000mL, obtains sample solution；Precision weighs D101 macroporous absorbent resin and is packed into chromatographic column, distilled water punching
Be washed till chromatographic column lower end trickle it is limpid it is colourless after, sample solution loading is adsorbed, it is to be adsorbed completely after with distilled water
Elution, then with ethanol elution, collects the ethanolic moiety of chromatographic column effluent, is concentrated, and dries, and grinding obtains Heiguteng exract active component
Cluster powder；Precision weighs Heiguteng exract active component cluster powder, with methanol constant volume, crosses 0.45 μm of miillpore filter, takes subsequent filtrate, obtain confession
Test sample solution；
(3) production of finger-print: chromatographic condition: chromatographic column: Xtimate C18, 4.6mm × 250mm, 5 μm；Mobile phase
For -0.1% phosphate aqueous solution (B) of acetonitrile (A)；Gradient elution, Detection wavelength 360nm, flow velocity 1mLmin-1, 38 DEG C of column temperature, into
10 μ L of sample amount；Record spectrogram obtains the HPLC finger-print of Heiguteng exract effective part group；
(4) confirmation of standard fingerprint spectrogram: according to the method for above-mentioned offer, multiple batches of Heiguteng exract effective part group is established
HPLC finger-print is compared by analysis and has been determined 24 shared peaks, these shared peaks constitute Heiguteng exract effective part group
Fingerprint characteristic, the standard finger-print as Heiguteng exract effective part group.
In the HPLC fingerprint image method for building up of Heiguteng exract effective part group above-mentioned, the mixed reference substance solution is made in this way
It is standby: to take neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid and 4,5-Dicaffeoylquinic acid standard items, with methanol constant volume be neochlorogenic acid, chlorogenic acid, hidden
Chlorogenic acid and 4,5-Dicaffeoylquinic acid concentration are 0.01mgmL-1Mixed reference substance solution.
In the HPLC fingerprint image method for building up of Heiguteng exract effective part group above-mentioned, the test solution is prepared:
Accurately weighed Heiguteng exract coarse powder 100.0g is extracted three times with 70% alcohol reflux, and the amount of alcohol of addition is respectively 8 times, 8 times, 6
Times, extraction time is respectively 1.5h, 1.5h, 1.0h, filtrate will merge three times, and filter, be concentrated into 180-220mL, use distilled water
It is settled to 1000mL, obtains sample solution；Precision weighs 100gD101 macroporous absorbent resin and is packed into chromatographic column, distilled water flushing to layer
Analyse column lower end trickle it is limpid it is colourless after, 1000mL sample solution loading is adsorbed, it is to be adsorbed completely after with 1200mL
It distills water elution and collects 70% ethanolic moiety of chromatographic column effluent then with 2500mL70% ethanol elution, be concentrated, drying is ground
Mill, obtains Heiguteng exract active component cluster powder；Precision weighs Heiguteng exract active component cluster powder 50.0mg, extremely with 70% methanol constant volume
10mL crosses 0.45 μm of miillpore filter, takes subsequent filtrate, obtain test solution.
In the HPLC fingerprint image method for building up of Heiguteng exract effective part group above-mentioned, the gradient elution is: mobile phase
For -0.1% phosphate aqueous solution (B) of acetonitrile (A)；Gradient elution program is: 0~15min, 11%~11%A；15~30min,
11%~18%A；30~40min, 18%~18%A；40~50min, 18%~23%A；50~60min, 23%~25%
A；60~85min, 25%~32%A.
In the HPLC fingerprint image method for building up of Heiguteng exract effective part group above-mentioned, 24 shared peaks are green with No. 3 peaks
Ortho acid is referring to peak, relative retention time is respectively 0.570,0.595,1.000,1.110,1.587,1.851,1.981,
2.117、2.177、2.436、2.561、2.720、3.317、4.092、4.277、4.360、4.443、4.580、4.844、
4.886、5.730、5.879、6.169、6.424。
Inventor has carried out a large amount of experiment, is the research of detection method of the present invention below
Experimental example: the HPLC finger-print research of Heiguteng exract effective part group
1 Heiguteng exract active component HPLC determining fingerprint pattern condition
1.1 instruments and reagent
1 laboratory apparatus table of table
Reagent methanol, acetonitrile are chromatographically pure, and ethyl alcohol is medical ethanol, and phosphoric acid is that analysis is pure, and water is distilled water and Wahaha
Pure water separately has D-101 macroreticular resin etc..
The preparation of 1.2 test solution and control solution
1.2.1 prepared by reference substance solution
It is appropriate that precision weighs neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid, 4,5-Dicaffeoylquinic acid standard items, is concentration with methanol constant volume
For 0.01mgmL-1Standard solution.
1.2.2 the preparation of test solution
Accurately weighed Heiguteng exract coarse powder (Longgong Area, Guizhou) 100.0g is extracted three times, the ethyl alcohol of addition with 70% alcohol reflux
Amount is respectively 8 times, 8 times, 6 times, and extraction time is respectively 1.5h, 1.5h, 1.0h, filtrate will merge three times, and filter, be concentrated into about
200mL is settled to 1000mL with distilled water, obtains sample solution.Precision weighs 100gD101 macroporous absorbent resin and is packed into chromatographic column,
Distilled water flushing to chromatographic column lower end trickle it is limpid it is colourless after, 1000mL sample solution loading is adsorbed, it is to be adsorbed
70% ethyl alcohol portion of chromatographic column effluent is collected then with 2500mL70% ethanol elution with 1200mL distillation water elution after completely
Point, it is concentrated, dries, grinding obtains Heiguteng exract active component cluster powder.Precision weighs Heiguteng exract active component cluster powder 50.0mg,
With 70% methanol constant volume to 10mL, 0.45 μm of miillpore filter is crossed, subsequent filtrate is taken, obtains test solution.
1.3 chromatographic condition
Chromatographic column is Xtimate C18(4.6mm × 250mm, 5 μm)；Mobile phase is -0.1% phosphate aqueous solution of acetonitrile, ladder
Degree elution (being shown in Table 2), Detection wavelength 360nm, flow velocity 1mLmin-1, 38 DEG C of column temperature, 10 μ L of sample volume.
2 mobile phase elution requirement of table
Reference substance is investigated according to identical chromatographic conditions sample introduction, by retention time to each chromatographic peak and chromatographic behavior into
Row compares, and has pointed out 4 chromatographic peaks altogether, respectively No. 2 peaks (neochlorogenic acid), No. 3 peaks (chlorogenic acid), No. 4 peaks (Cryptochlorogenic acid),
No. 15 peaks (4,5-Dicaffeoylquinic acid).See Fig. 1 and Fig. 2:
2 methodological studies
It is to meet to investigate and prove that finger-print measurement evaluation method to be taken is reliable, repeatable
The condition of traditional Chinese medicine fingerprint measurement, has carried out the verifying of methodology to the method for experiment for this.Fingerprint map analyzing process is multiple
Miscellaneous, influence factor is more, it is therefore desirable to carry out methodology validation experiment and verify to measurement result overall process, be the quality of medicinal material
Control provides reliable data information and process analysis procedure analysis control node, and this respect needs give abundant consideration when making a search.
In consideration of it, this part has carried out methodological study to the precision, repeatability and stability of experimental method.
2.1 Precision Experiment
Precision weighs Heiguteng exract active component cluster powder (Longgong Area, Guizhou) 50.0mg, with 70% methanol constant volume to 10mL, mistake
0.45 μm of miillpore filter, takes subsequent filtrate, obtains test solution, by chromatographic condition continuous sample introduction 6 times under " 1.3 " item, calculates each total
There is the RSD of peak relative retention time and relative peak area to be respectively less than 3.0%, shows that instrument precision is good.It is shown in Table 3, table 4, table 5
With table 6.
3 precision test retention time of table
4 precision test peak area of table
5 precision test relative retention time of table
6 precision test relative peak area of table
2.2 stability experiment
Precision weighs Heiguteng exract active component cluster powder (Longgong Area, Guizhou) 50.0mg, with 70% methanol constant volume to 10mL, mistake
0.45 μm of miillpore filter, takes subsequent filtrate, obtains test solution, by chromatographic condition under " 1.3 " item respectively in 0,2,4,8,24,36h
Sample introduction, the RSD of the relative retention time and relative peak area that calculate each shared peak are respectively less than 3.0%, and experiment shows test sample
Solution is stablized in 36h at normal temperature.It is shown in Table 7, table 8, table 9 and table 10.
7 stability test retention time of table
8 stability test peak area of table
9 stability test relative retention time of table
10 stability test relative peak area of table
2.3 repeated experiment
Precision weighs Heiguteng exract active component cluster powder (Longgong Area, Guizhou) 50.0mg, and 6 parts, extremely with 70% methanol constant volume
10mL crosses 0.45 μm of miillpore filter, takes subsequent filtrate, obtain test solution, is analyzed respectively by chromatographic condition under " 1.3 " item,
The RSD of the relative retention time and relative peak area that calculate each shared chromatographic peak is respectively less than 3.0%, shows that method used repeats
Property is preferable.It is shown in Table 11, table 12, table 13 and table 14.
11 repetitive test retention time of table
12 repetitive test peak area of table
13 repetitive test relative retention time of table
14 repetitive test relative peak area of table
The foundation of 3 Heiguteng exract effective part group HPLC finger-prints
To establish Heiguteng exract active component group fingerprint atlas, the Heiguteng exract of 11 batches of separate sources is acquired altogether, is identified as black
Bone rattan (Periploca forrestii Schltr.), respectively number be S1, S2, S3, S4, S5, S6, S7, S8, S9, S10,
S11.Sample detailed derivation is shown in Table 15.Precision weighs the Heiguteng exract medicinal powder 100.0g of 11 batches of separate sources, according to " 1.2.2 "
Legal system available test agent solution below by chromatographic condition sample introduction under " 1.3 " item and records chromatogram.By to 11 batches of sample colors
Spectrum analysis, indicates 24 shared peaks altogether, and 11 batches of Heiguteng exract effective part groups share peak and see Fig. 3-Fig. 5, pair of common pattern
Fig. 2 is seen according to finger-print.Each shared peak retention time of the active component group fingerprint atlas of 11 batches of Heiguteng exract samples and peak area are shown in
Table 16 and table 18, with No. 3 peaks (chlorogenic acid is referring to peak), each shared peak of the active component group fingerprint atlas of 11 batches of Heiguteng exract samples
Relative retention time and relative peak area are shown in Table 17 and 19.
15 11 batches of separate sources Heiguteng exract sample messages of table
16 11 batches of each shared peak retention times of Heiguteng exract sample active component group fingerprint atlas of table
17 11 batches of each shared peak relative retention times of Heiguteng exract sample active component group fingerprint atlas of table
18 11 batches of each shared peak peak areas of Heiguteng exract sample active component group fingerprint atlas of table
19 11 batches of each shared peak relative peak areas of Heiguteng exract sample active component group fingerprint atlas of table
3.1 Heiguteng exract effective part group fingerprint similarities
Using Chinese Pharmacopoeia Commission's similarity evaluation software with the chromatic graph spectrum of S1 sample for referring to map, average is raw
Cheng Fa, " time window width " are 0.1min, and Auto-matching is carried out after Supplements, generate control map, obtain each place of production black bone
Rattan effective part group HPLC fingerprint similarity, is shown in Table 20.11 batches of Heiguteng exract effective part group similarities 0.837~
Range between 0.989, wherein there is the Heiguteng exract effective part group similarity value in 3 batches of places of production 0.9 hereinafter, remaining 8 batches of Heiguteng exract
The similarity of effective part group is 0.9 or more.
20 11 batches of Heiguteng exract sample HPLC fingerprint similarities of table
The calibration at 3.2 shared peaks
1, reference substance source and lot number are shown in Table 21.
21 reference substance source of table and lot number
2, above-mentioned each reference substance is investigated according to identical chromatographic conditions sample introduction, passes through the retention time and color to each chromatographic peak
Spectrum behavior is compared, and has pointed out 4 chromatographic peaks altogether, and respectively No. 2 peaks (neochlorogenic acid), No. 3 peaks (chlorogenic acid), No. 4 peaks are (hidden
Chlorogenic acid), No. 15 peaks (4,5-Dicaffeoylquinic acid).See Fig. 5 and Fig. 6.
4 results and conclusion
The optimizing research of 1 chromatographic condition
The determination of 1.1 mobile phases
Chemical component quantity that the chromatographic condition of finger-print will be such that chromatogram reflects in medicinal material as much as possible and its at
Divide ratio, to obtain the characteristic fingerprint pattern of Heiguteng exract effective part group, on the basis of early-stage study, to following four groups of mobile phases
It is compared selection:
First group: -0.1% phosphate aqueous solution of acetonitrile is measured by different gradients.
Second group: -0.1% phosphate aqueous solution of tetrahydrofuran is measured by different gradients.
Third group: -0.1% phosphate aqueous solution of methanol is measured by different gradients.
4th group: -0.2% phosphate aqueous solution of methanol is measured by different gradients.
By chromatogram it is found that using -0.1% phosphate aqueous solution of acetonitrile as mobile phase, depth-graded elution effect is best, color
Chromatographic peak in spectrogram is more, and separating degree is preferable, and retention time is moderate, so select -0.1% phosphate aqueous solution of acetonitrile as stream
Dynamic phase.
The investigation of 1.2 Heiguteng exract effective part group HPLC finger-print column temperatures
More preferably column temperature is also investigated in chromatogram, this experiment in order to obtain, and column temperature is respectively 30 DEG C, 35 DEG C, 38
DEG C, 40 DEG C, by being compared to chromatogram, it is known that chromatographic peak separating degree is best when column temperature is 38 DEG C, therefore the final choosing of this research
Fixed column temperature is 38 DEG C.
The selection of 1.3 Detection wavelengths
This experimental selection 220,254,274,360nm tetra- wavelength are measured, the chromatographic peak occurred at 360 nm compared with
It is more, and most of chromatographic peak absorption is stronger, therefore this experimental selection 360nm is as Detection wavelength.
The investigation of 1.4 test sample solvents
It is that 60% methanol, 70% methanol, 80% methanol are investigated respectively to test sample solvent, it can from obtained chromatogram
Know, when solvent is 70% methanol, the peak area of each main chromatographic peak no longer has an impact with the increase of solvent usage later, so
This experiment finally determines that test sample solvent strength is 70% methanol.
Experiments have shown that, the measuring method is reliable and stable, and finger-print is relatively stable above, and can be used for controlling Heiguteng exract has
Imitate the quality of part group.
Compared with prior art, this method has easy, stable, precision height, high repeatability and other advantages；Also with this method
It more can comprehensively reflect the type and quantity of contained chemical component in Heiguteng exract effective part group, and then its quality be carried out whole
Body description and evaluation.And the Heiguteng exract active component group fingerprint atlas chemical component that is detected with this method is relatively more, each spy
Sign peak heights ratio is moderate, and baseline is more steady, and separating degree, peak shape and column are imitated.
Detailed description of the invention
Fig. 1 is reference substance efficient liquid phase experiment chromatogram；
Fig. 2 is Heiguteng exract sample efficient liquid phase experiment chromatogram；
Fig. 3 is 11 batches of Heiguteng exract effective part group HPLC finger-prints；
Fig. 4 is 11 batches of Heiguteng exract effective part group HPLC control maps；
Fig. 5 is the HPLC map for mixing reference substance；
Fig. 6 is the reference fingerprint of common pattern.
Below with reference to embodiment, the present invention is further illustrated.
Specific embodiment
Embodiment 1:
A kind of method for building up of the HPLC fingerprint image of Heiguteng exract effective part group, comprising the following steps:
A kind of HPLC fingerprint image method for building up of Heiguteng exract effective part group, comprising the following steps:
(1) preparation of mixed reference substance solution: taking neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid and 4,5-Dicaffeoylquinic acid standard items,
It is neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid and 4,5-Dicaffeoylquinic acid concentration with methanol constant volume is 0.01mgmL-1Mix reference substance
Solution；
(2) preparation of test solution: accurately weighed Heiguteng exract coarse powder 100.0g is extracted three times with 70% alcohol reflux,
The amount of alcohol of addition is respectively 8 times, 8 times, 6 times, and extraction time is respectively 1.5h, 1.5h, 1.0h, filtrate will merge three times, and take out
Filter, is concentrated into 180-220mL, is settled to 1000mL with distilled water, obtains sample solution；Precision weighs 100gD101 macroporous absorption tree
Rouge be packed into chromatographic column, distilled water flushing to chromatographic column lower end trickle it is limpid it is colourless after, by 1000mL sample solution loading into
Row absorption, it is to be adsorbed completely after water elution distilled with 1200mL, then with 2500mL70% ethanol elution, collect chromatographic column effluent
70% ethanolic moiety, be concentrated, dry, grinding, obtain Heiguteng exract active component cluster powder；Precision weighs Heiguteng exract effective part group
Powder 50.0mg is crossed 0.45 μm of miillpore filter, is taken subsequent filtrate, obtain test solution with 70% methanol constant volume to 10mL；
(3) production of finger-print: chromatographic condition: chromatographic column: Xtimate C18, 4.6mm × 250mm, 5 μm；Mobile phase
For -0.1% phosphate aqueous solution (B) of acetonitrile (A)；Gradient elution, gradient elution program are: 0~15min, 11%~11%A；15
~30min, 11%~18%A；30~40min, 18%~18%A；40~50min, 18%~23%A；50~60min,
23%~25%A；60~85min, 25%~32%A.Detection wavelength 360nm, flow velocity 1mLmin-1, 38 DEG C of column temperature, sample volume
10μL；Record spectrogram obtains the HPLC finger-print of Heiguteng exract effective part group；
(4) confirmation of standard fingerprint spectrogram: according to the method for above-mentioned offer, multiple batches of Heiguteng exract effective part group is established
HPLC finger-print is compared by analysis and has been determined 24 shared peaks, these shared peaks constitute Heiguteng exract effective part group
Fingerprint characteristic, the standard finger-print as Heiguteng exract effective part group.24 shared peaks are with No. 3 peak chlorogenic acids
Referring to peak, relative retention time is respectively 0.570,0.595,1.000,1.110,1.587,1.851,1.981,2.117,
2.177、2.436、2.561、2.720、3.317、4.092、4.277、4.360、4.443、4.580、4.844、4.886、
5.730、5.879、6.169、6.424。
Claims (5)
1. a kind of HPLC fingerprint image method for building up of Heiguteng exract effective part group, it is characterised in that: the following steps are included:
(1) preparation of mixed reference substance solution: neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid and 4,5-Dicaffeoylquinic acid standard items are taken, first is used
Alcohol constant volume is that neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid and 4,5-Dicaffeoylquinic acid concentration are 0.005-0.015gmL-1Mixing control
Product solution；
(2) preparation of test solution: accurately weighed Heiguteng exract coarse powder 100.0g is extracted with alcohol reflux, is filtered, and is concentrated, and is used
Distilled water constant volume 1000mL, obtains sample solution；Precision weighs D101 macroporous absorbent resin and is packed into chromatographic column, distilled water flushing to layer
Analyse column lower end trickle it is limpid it is colourless after, sample solution loading is adsorbed, it is to be adsorbed completely after to distill water elution, so
Afterwards with ethanol elution, the ethanolic moiety of chromatographic column effluent is collected, is concentrated, is dried, grinding obtains Heiguteng exract active component cluster powder；
Precision weighs Heiguteng exract active component cluster powder, with methanol constant volume, crosses 0.45 μm of miillpore filter, takes subsequent filtrate, it is molten to obtain test sample
Liquid；
(3) production of finger-print: chromatographic condition: chromatographic column: XtimateC18, 4.6 × 250mm, 5 μm；Mobile phase: acetonitrile A
Phase, 0.1% phosphate aqueous solution are B phase；Gradient elution, Detection wavelength 360nm, flow velocity 1mLmin-1, 38 DEG C of column temperature, into
10 μ L of sample amount；Record chromatogram obtains the HPLC finger-print of Heiguteng exract effective part group；
(4) confirmation of standard fingerprint spectrogram: according to the method for above-mentioned offer, multiple batches of Heiguteng exract effective part group is established
HPLC finger-print is compared by analysis and 24 shared peaks has been determined, these shared peaks constitute Heiguteng exract effective part group
Fingerprint characteristic, the standard finger-print as Heiguteng exract effective part group.
2. the HPLC fingerprint image method for building up of Heiguteng exract effective part group as described in claim 1, it is characterised in that: described mixed
It closes reference substance solution to be prepared: taking neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid and 4,5-Dicaffeoylquinic acid standard items, be with methanol constant volume
Neochlorogenic acid, chlorogenic acid, Cryptochlorogenic acid and 4,5-Dicaffeoylquinic acid concentration are 0.01mgmL-1Mixed reference substance solution.
3. the HPLC fingerprint image method for building up of Heiguteng exract effective part group as described in claim 1, it is characterised in that: the confession
Test sample solution is prepared: accurately weighed Heiguteng exract coarse powder 100.0g, is extracted three times with 70% alcohol reflux, the amount of alcohol of addition
Respectively 8 times, 8 times, 6 times, extraction time are respectively 1.5h, 1.5h, 1.0h, filtrate will merge three times, and filter, be concentrated into 180-
220mL is settled to 1000mL with distilled water, obtains sample solution；Precision weighs 100gD101 macroporous absorbent resin and is packed into chromatographic column,
Distilled water flushing to chromatographic column lower end trickle it is limpid it is colourless after, 1000mL sample solution loading is adsorbed, it is to be adsorbed
70% ethyl alcohol portion of chromatographic column effluent is collected then with 2500mL70% ethanol elution with 1200mL distillation water elution after completely
Point, it is concentrated, dries, grinding obtains Heiguteng exract active component cluster powder；Precision weighs Heiguteng exract active component cluster powder 50.0mg,
With 70% methanol constant volume to 10mL, 0.45 μm of miillpore filter is crossed, subsequent filtrate is taken, obtains test solution.
4. the HPLC fingerprint image method for building up of Heiguteng exract effective part group as described in claim 1, it is characterised in that: described
Gradient elution program is: 0~15min, 11%~11%A；15~30min, 11%~18%A；30~40min, 18%~
18%A；40~50min, 18%~23%A；50~60min, 23%~25%A；60~85min, 25%~32%A.
5. the HPLC fingerprint image method for building up of Heiguteng exract effective part group as described in claim 1, it is characterised in that: described 24
A shared peak is referring to peak with No. 3 peak chlorogenic acids, relative retention time is respectively 0.570,0.595,1.000,1.110,
1.587、1.851、1.981、2.117、2.177、2.436、2.561、2.720、3.317、4.092、4.277、4.360、
4.443、4.580、4.844、4.886、5.730、5.879、6.169、6.424。
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| CN110702813A (en) * | 2019-10-23 | 2020-01-17 | 贵州中医药大学 | Miao medicine caulis et folium periplocae HPLC fingerprint spectrum research and multi-component content determination method |
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| CN110702813A (en) * | 2019-10-23 | 2020-01-17 | 贵州中医药大学 | Miao medicine caulis et folium periplocae HPLC fingerprint spectrum research and multi-component content determination method |
| CN110702813B (en) * | 2019-10-23 | 2022-03-11 | 贵州中医药大学 | Miao medicine caulis et folium periplocae HPLC fingerprint spectrum research and multi-component content determination method |
| CN114166954A (en) * | 2021-04-07 | 2022-03-11 | 国药集团同济堂（贵州）制药有限公司 | Preparation method and quality detection method of caulis et folium Periplocae Forrestii standard decoction |
| CN114166954B (en) * | 2021-04-07 | 2023-10-17 | 国药集团同济堂（贵州）制药有限公司 | Preparation method and quality detection method of black bone vine standard decoction |
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