CN104931620B - The separation of Gynura procumbens (Lour.) Merr. Content of Chlorogenic Acid constituents and content assaying method - Google Patents
The separation of Gynura procumbens (Lour.) Merr. Content of Chlorogenic Acid constituents and content assaying method Download PDFInfo
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- dicaffeoylquinic
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- 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 title claims abstract description 86
- 235000001368 chlorogenic acid Nutrition 0.000 title claims abstract description 53
- 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 title claims abstract description 51
- 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 title claims abstract description 51
- 229940074393 chlorogenic acid Drugs 0.000 title claims abstract description 51
- 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 title claims abstract description 51
- 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 title claims abstract description 51
- 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 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 37
- 240000008672 Gynura procumbens Species 0.000 title claims abstract description 32
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- 238000000926 separation method Methods 0.000 title description 6
- 239000000470 constituent Substances 0.000 title description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 54
- 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 abstract description 47
- 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 abstract description 46
- 239000013558 reference substance Substances 0.000 claims abstract description 31
- 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 abstract description 28
- 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 abstract description 28
- 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 abstract description 25
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- 239000000523 sample Substances 0.000 claims abstract description 25
- KRZBCHWVBQOTNZ-PSEXTPKNSA-N 3,5-di-O-caffeoyl quinic acid Chemical compound O([C@@H]1C[C@](O)(C[C@H]([C@@H]1O)OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)C(O)=O)C(=O)\C=C\C1=CC=C(O)C(O)=C1 KRZBCHWVBQOTNZ-PSEXTPKNSA-N 0.000 claims abstract description 24
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
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- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 claims description 8
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
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- CWVRJTMFETXNAD-BMNNCGMMSA-N (1s,3r,4s,5r)-3-[(e)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy-1,4,5-trihydroxycyclohexane-1-carboxylic 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-BMNNCGMMSA-N 0.000 abstract description 7
- KRZBCHWVBQOTNZ-DLDRDHNVSA-N isochlorogenic acid Natural products O[C@@H]1[C@H](C[C@@](O)(C[C@H]1OC(=O)C=Cc2ccc(O)c(O)c2)C(=O)O)OC(=O)C=Cc3ccc(O)c(O)c3 KRZBCHWVBQOTNZ-DLDRDHNVSA-N 0.000 abstract description 7
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- XXLFLUJXWKXUGS-UHFFFAOYSA-N 6-methoxyquinoline-4-carboxylic acid Chemical compound N1=CC=C(C(O)=O)C2=CC(OC)=CC=C21 XXLFLUJXWKXUGS-UHFFFAOYSA-N 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
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- QAIPRVGONGVQAS-DUXPYHPUSA-N trans-caffeic acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-DUXPYHPUSA-N 0.000 description 2
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- ACEAELOMUCBPJP-UHFFFAOYSA-N (E)-3,4,5-trihydroxycinnamic acid Natural products OC(=O)C=CC1=CC(O)=C(O)C(O)=C1 ACEAELOMUCBPJP-UHFFFAOYSA-N 0.000 description 1
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- LOUPRKONTZGTKE-WZBLMQSHSA-N Quinine Chemical class C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-WZBLMQSHSA-N 0.000 description 1
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Abstract
The invention discloses the method for quantitatively determining of two kinds of Gynura procumbens (Lour.) Merr. extractum Content of Chlorogenic Acids, neochlorogenic acid and three kinds of isochlorogenic acid.Step is as follows：A, determine optimum chromatogram condition；B, take neochlorogenic acid, chlorogenic acid, three kinds of isochlorogenic acid reference substances, plus methanol makes reference substance solution；C, take Gynura procumbens (Lour.) Merr. extractum, plus methanol, filter, obtain sample；D, respectively accurate absorption reference substance and sample solution, chromatography column feed materials, measure the content of chlorogenic acid, neochlorogenic acid and three kinds of isochlorogenic acid；Chlorogenic acid reference substance solution is only prepared, with chlorogenic acid for comparison, 4,5-Dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, neochlorogenic acid relative correction factor are respectively 1.21,1.12,1.07,0.92, measure the content of five kinds of materials in e or b.The present invention sets up itself f and other compositions between with chlorogenic acid as referencek/s, and calculate the amount of each composition, external standard and survey comment method accuracy and reliability quite, this provides new mode for more authentic assessment Gynura procumbens (Lour.) Merr. quality.
Description
Technical field
The present invention relates to Chinese herbal medicine field of quality control is and in particular to the quality determining method of Gynura procumbens (Lour.) Merr. extractum, more
In particular it relates to the method for quantitatively determining of Gynura procumbens (Lour.) Merr. extractum Content of Chlorogenic Acid, neochlorogenic acid and three kinds of isochlorogenic acid.
Background technology
Gynura procumbens (Lour.) Merr. (Gynura procumbens (Lour.) Merr) is commonly called as handkerchief and collapses plate, be Compositae Gynura Cass for many years
Raw herbaceous plant, dietotherapeutic, there is extensive pharmacological action,《Dai medicine》Record handkerchief and collapse plate " dissipating blood stasis, detumescence, promoting blood circulation life
Flesh ", in the Southeast Asia ethnic mimority area of China's periphery, it is additionally operable to hypoglycemic, anti-liver cancer and anti-, solution dysentery and protection intestinal.Among the people
Often young stem and leaf is made vegetable food, makees tea-drinking, as new raw-food material (former Ministry of Public Health announce 2012 No. 8), it is in Guangdong, river
There is the plantation of scale on the ground such as west.Known composition include chlorogenic acid (graceful Liao of Zhu Yu be bright, Zheng Guodong, Zhang Qingfeng. Gynura procumbens (Lour.) Merr.
Content of Chlorogenic Acid extract and purifying process optimization. hubei agricultural science .2012,51 (19) .), caffeic acid, flavone, alkaloid, terpene
Alkene, Coumarinses etc..Existing document reports extraction and the purifying process of its Content of Chlorogenic Acid, in Gynura procumbens (Lour.) Merr., other are green
The research of ortho acid analogous components not yet relates to, and has no the relevant report of its quality standard at present, and therefore, applicant separates and makes
Standby each composition, sets up HPLC method simultaneously and measures each component content.
Need using traditional external standard method multi objective that the species of reference substance is many, quantity is big, expensive, have document to propose
(He Bing, Liu Yan, Yang Shiyan, a swallow .HPLC mono- surveys and comments method to measure double blue or green pharynxs more simultaneously more for the one Quality Control pattern surveying the multi objective commented
10 kinds of compositions in lozenge. Chinese herbal medicine, 2013,08 (44):974-981.), with reference substance cheap and easy to get as internal standard, with this composition
Relative correction factor (f with other compositionsk/s), to calculate the amount of other compositions.This experiment, with chlorogenic acid as object of reference, is set up
Itself f and other compositions betweenk/s, and calculate the content of each composition, contrasted with the result using external standard method, to be verified one
Survey the accuracys commented and reliability more.This provides new mode for more authentic assessment Gynura procumbens (Lour.) Merr. quality.
Content of the invention
For the deficiencies in the prior art, the purpose of the present invention first consists in and provides a kind of separation preparation prostration chrysanthemum
The HPLC method of Radix Notoginseng Content of Chlorogenic Acid, neochlorogenic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid and 4,5-Dicaffeoylquinic acid；
Based on above-mentioned HPLC method, invention further provides two kinds of Gynura procumbens (Lour.) Merr. Content of Chlorogenic Acids, neochlorogenic acid and
The method for quantitatively determining of three kinds of isochlorogenic acid.
To achieve these goals, present invention employs following technical measures：
Present invention monomer component in preparative high performance liquid chromatography separation Gynura procumbens (Lour.) Merr., using LC-MS, core
Pumping Magnetic Resonance Method identifies its structure.Set up the HPLC method of energy its component content of Accurate Determining, isolate and purify from Gynura procumbens (Lour.) Merr.
Chlorogenic acid, neochlorogenic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid and 4,5-Dicaffeoylquinic acid, wherein 3,5-Dicaffeoylquinic acid, B and C plant from this genus first
Separate in thing.On this basis, with chlorogenic acid for comparison, calculate relative correction factor, set up " one surveys comment " method, relative correction more
The good tolerance of the factor, one surveys to comment method and external standard method comparing result no significant difference more.The content assaying method set up can
For measuring 5 kinds of chlorogenic acid compositions in Gynura procumbens (Lour.) Merr. extract simultaneously.
In preparation system, applicant compared for acetonitrile-water and acetonitrile -0.1v/v% trifluoroacetic acid aqueous solution system, knot
Fruit adopts acetonitrile -0.1v/v% trifluoroacetic acid aqueous solution elution system in the case of high concentration sample introduction, and eluting separating effect is best,
Theoretical cam curve highest, saves cost；In analysis system, acetonitrile -0.1v/v% trifluoroacetic acid aqueous solution eluent system is not
Can make to efficiently separate between 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid and 4,5-Dicaffeoylquinic acid, and use the acetonitrile -0.1w/v% (unit of w/v
It is g/mL) phosphate aqueous solution eluent system makes each monomer separation degree high.Compare ultra-pure water, 98v/v% methanol, 50v/ simultaneously
Three kinds of extraction solvents of v% methanol, the methanol effect of result 50v/v% is more excellent.
Individually measure component content in medical material take, effort, inefficiency, high cost, can not meet Chinese crude drug quality
The demand of research, the method simultaneously measuring Multiple components content becomes one of effective ways of quality control, and the present invention is set up
Condition be that the separation of the extremely similar composition of chlorogenic acid provides reference.
Application with Gynura procumbens (Lour.) Merr. progressively increases, and only controls the content of its Content of Chlorogenic Acid obviously can not truly evaluate
The inherent quality of medicine, and survey to comment method using one more, in range of liner, the amount of composition is directly proportional to detector response,
Measure the content of other four kinds of compositions by using a kind of common reference substance simultaneously, can comprehensively and truly evaluate Gynura procumbens (Lour.) Merr.
Quality.
Brief description
Fig. 1 is the HPLC collection of illustrative plates obtained by step 1.3 in embodiment 1, and wherein, 1 is neochlorogenic acid；2 is chlorogenic acid；3 are
3,4-Dicaffeoylquinic acid；4 is 3,5-Dicaffeoylquinic acid；5 is 4,5-Dicaffeoylquinic acid.
Fig. 2 is the HPLC chromatogram of the mixing reference substance (A) in embodiment 2 and sample (B).
Specific embodiment
Applicant will be described in detail to the inventive method in conjunction with specific embodiments below.
The isolation identification of embodiment 1 Gynura procumbens (Lour.) Merr. Content of Chlorogenic Acid constituents
The preparation of 1 Gynura procumbens (Lour.) Merr. extractum sample
1.1 extractum sample preparation Gynura procumbens (Lour.) Merr. originate from Guangdong, are identified by Chinese Academy of Sciences plants in Wuhan Botanical Garden teacher Zhang Ping Kun.Take
Stem (0.5) is dried pulverize, adds the 80v/v% ethanol of 5L, reflux, extract, 1h at 85 DEG C every time, extract 3 times altogether, united extraction
Liquid concentrate drying, extractum (density 1.40g/L) yield is 18.5%.Extractum is dissolved in methanol (extractum and the 50v/ of 50v/v%
The ratio of the methanol of v% is 0.1g：1mL), with 0.45 μm of filtering with microporous membrane, so that preparation uses.
Again by above-mentioned experiment repetitive operation 3 times, four batch extractum are obtained, lot number is respectively 2012/6/14,2012/6/20,
2012/6/21,2012/9/6 uses for subsequent experimental.
1.2 preparative hplies and Mass Spectrometry Conditions
1.2.1 chromatographic condition：DIONEX UltiMate 3000 high performance liquid chromatograph, chromatographic column is WATERS C18-
MS- II chromatographic column (10 × 250mm, 5 μm)；Mobile phase is acetonitrile (A) -0.1v/v% trifluoroacetic acid (B), gradient elution：0～
40min, 5%～38%A (v/v%), 40～45min, 38%～5%A (v/v%), flow rate of mobile phase 3.0mL/min, sample introduction
Measure 500 μ L, Detection wavelength 326nm；Column temperature：30℃.
1.2.2 Mass Spectrometry Conditions：320 DEG C of capillary temperature, nebulizer pressure 20Pa, source voltage 5KV, total current 100 μ A, does
320 DEG C of dry device temperature, dry gas stream speed 10L/min, detection mode is MSn(n=1～3), full scan mass spectrum.Mass scanning model
Enclose M/Z 100～600.
1.3 isolate and purify process
Sample introduction under 1.2.1 chromatographic condition, chromatogram is shown in Fig. 1, collects the fraction at 1～No. 9 peak, through rotary evaporation, freezes
Dry, obtain 9 kinds of brown ceramic powders, No. 2 sample sizes>50mg, No. 3 sample sizes are 5mg, No. 4 sample 54mg, No. 5 sample 32mg, other
It is below 1mg.
1.4 chlorogenic acid compound structure identifications
Compound 3, No. 4, No. 5, be light brown powder, three1H-NMR、13C-NMR peak-to-peak signal is extremely similar, should be
Isomerss, [M-H]?The m/z of ion is 517, [M-H]?The MS of ion2The larger ion m/z of abundance is 353,191,179
With 135,1H-NMR (400MHz, in CH3DO have in) two groups of trans olefins signals [δ 7.50 (1H, d, J=16.0Hz), 7.46
(1H, d, J=16.0Hz), 6.25 (1H, d, J=16.0Hz), 6.15 (1H, d, J=16.0Hz)], exist and exist with quininic acid
Characteristic signal [δ 7.04 (2H, brd, J=1.9Hz), 6.99 (2H, brd, J=that 1,3,5- trisubstituted phenyl ring proton has
8.1Hz), 6.78 (2H, brd, J=8.0,1.9Hz)]；Compound 4 has quininic acid H-3, and H-5 is in δ 5.21 (2H, m) feature letter
Number, in δ 3.84 (1H, brd) the diagnostic protons signal of High-Field relatively, in quininic acid structure, the hydrogen signal of two methylene exists H-4
High-Field position δ 2.13 (2H), 2.14 (2H) have characteristic signal.And compound 5, the characteristic signal of quininic acid H-3, H-4 is in δ 4.98
(2H), 5.33 (2H), and the proton signal of H-5, in low field position δ 4.18 (1H, brd) relatively, can speculate the two all in conjunction with carbon spectrum
For two coffee quinine acids thus it is speculated that compound molecule formula is C25H24O12, calculating degree of unsaturation is 14.By with document nuclear-magnetism number
According to contrast it is known that No. 4 samples are 3,5-Dicaffeoylquinic acid (3,5- dicaffeoyl-quinic acid), No. 3, No. 4, the carbon spectrum of No. 5, hydrogen spectrum, mass spectrum
Figure is all extremely similar, and No. 3 is 3,4-Dicaffeoylquinic acid (3,4- bis- coffee quininic acid), and No. 5 samples are 4,5-Dicaffeoylquinic acid (4,5- bis- coffee quinines
Acid).
No. 1 peak and No. 2 peaks, in its MS spectrum, [M-H]?M/z be 353, MS2In spectrum, the larger quasi-molecular ions of abundance for m/z is
191st, 164,135, wherein 191 is quininic acid fragment ion peak, and 164 is caffeic acid fragment ion peak, and 135 take off one point for caffeic acid
Fragment ion peak after sub- water, both are isomerss, and by can determine whether with standard substance contrast, No. 2 higher chromatographic peaks are green
Ortho acid, No. 1 is neochlorogenic acid.
The method of embodiment 2 external standard method chlorogenic acid, neochlorogenic acid and three kinds of isochlorogenic acid contents
2.1 analysis chromatographic conditions
DIONEX UltiMate 3000 high performance liquid chromatograph；DIONEX C18 chromatographic column (250 × 4.6mm, 5 μm),
Mobile phase is acetonitrile (A) -0.1w/v% phosphate aqueous solution (B) (unit of w/v is g/mL), gradient elution：0～65min, 9%
～27%A (v/v%)；65～70min, 27%～9% (v/v%) A；Time is 70min, volume flow 1.0mL/min, sample introduction
Measure 10 μ L, Detection wavelength 326nm, 30 DEG C of column temperature.
2.2 solution preparations
2.2.1 sample preparation
Precision weighs extractum 100-250mg in embodiment 1, in 10mL volumetric flask, plus 9mL 50v/v% methanol, Yu Chao
In sound instrument, the ultrasonic 15-30min of 20-40KHz, lets cool, plus 50v/v% methanol shakes up to scale, crosses 0.45 μm of filter membrane, obtains final product sample
Solution.
2.2.2 mixed reference substance solution preparation
Gynura procumbens (Lour.) Merr. extractum (2012/6/ is recorded using Chinese version annex VIII M aquametry the first method A in 2010
14), chlorogenic acid reference substance, neochlorogenic acid reference substance, 3,5-Dicaffeoylquinic acid reference substance, 3,4-Dicaffeoylquinic acid reference substance, 4,5-Dicaffeoylquinic acid comparison
Product water content is respectively 9.46%, 6%, 5.85%, 11.94%, 13.53%, 6.12%.
Weigh neochlorogenic acid, chlorogenic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid reference substance 3.30,6.12,
5.96th, 6.56,7.88mg is placed in 10mL volumetric flask, adds 50v/v% methanol to dissolve and be diluted to scale, shakes up, make matter
Amount concentration be respectively 0.31,0.57,0.58,0.63, the mixed reference substance solution of 0.74mg/mL, cross 0.45 μm of microporous filter membrane standby
With.
2.2.3 system suitability experiment
Be analyzed under 2.1 chromatographic conditions, mixed reference substance solution and sample solution (2012/9/6,2012/6/21,
2012/6/20th, 2012/6/14 collection of illustrative plates is no clearly distinguished from, therefore with 2012/6/14 collection of illustrative plates as representative) result see respectively Fig. 2A and
Fig. 2 B.The separating degree of the chromatographic peak that chlorogenic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid are adjacent is all higher than 1.5, drags
, between 1.00-1.20, in terms of each chromatographic peak all more than 10000, neochlorogenic acid is excessive because of polarity for theoretical cam curve for the tail factor,
Separating effect is slightly worse, but because its content is few relative to other compositions, therefore do not optimize further.
2.2.4 linear relationship is investigated
Precision measure mixed reference substance solution 0.1,0.2,0.4,0.6,0.8,1.0,2.0mL, be respectively placed in 10mL capacity
In bottle, plus 50v/v% methanol constant volume, to scale, shakes up, 0.45 μm of filtering with microporous membrane, sample introduction under 2.1 chromatographic conditions, record
Chromatographic peak area.With reference substance mass concentration as vertical coordinate (Y), with peak area as abscissa (X), draw standard curve, carry out
Linear regression, regression equation and the range of linearity are shown in Table 1.Result shows, each reference substance linearly closes in the range of respective mass concentration
System is good.
Result investigated by table 1 linear relationship
2.2.5 Precision Experiment
The accurate mixed reference substance solution drawing 2.2.2 preparation, repeats sample introduction 6 times, sampling volume under 2.1 chromatographic conditions
For 5 or 10 μ L, with reference substance calculated by peak area, neochlorogenic acid, chlorogenic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid peak
The RSD of area is respectively 3.21%0.06%, 0.18%, 0.14%, 0.08%.
2.2.6 stability experiment
Take sample solution (2012/9/6) respectively in 0,2,4,6,12,24 hours sample introductions, record neochlorogenic acid, chlorogenic acid,
3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid peak area RSD be respectively 5.51%, 0.30%, 0.35%, 1.75%,
0.73%, show that sample is stable in 24 hours.
2.2.7 repeated experiment
Take same batch Gynura procumbens (Lour.) Merr. extractum (be all prepared by embodiment 1 1.1 method, (2012/6/21) about
500mg, prepares 6 parts of sample solution, records neochlorogenic acid, chlorogenic acid, 3,5-Dicaffeoylquinic acid, isochlorogenic acid respectively by parallel weighed 6 parts
B, the RSD of 4,5-Dicaffeoylquinic acid mass fraction are respectively 3.78%, 1.40%, 2.76%, 2.11%, 2.07%.
2.2.8 average recovery experiment
Take and measure neochlorogenic acid, chlorogenic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, the Gynura procumbens (Lour.) Merr. of 4,5-Dicaffeoylquinic acid content
Extractum (2012/6/21), accurately weighed, every part is simultaneously introduced a certain amount of neochlorogenic acid, chlorogenic acid, 3,5-Dicaffeoylquinic acid, different green former
Sour B, 4,5-Dicaffeoylquinic acid reference substance, prepare 9 parts of sample solution, under 2.1 chromatographic conditions, neochlorogenic acid, chlorogenic acid, isochlorogenic acid
A, 3,4-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid average recovery rate are respectively 109.9%, 101.36%, 93.9%, 104.2%, 105.9%,
RSD is respectively 1.12%, 0.59%, 1.03%, 1.22%, 1.01%.
2.2.9 sample size measures
Take the Gynura procumbens (Lour.) Merr. extractum of different lot numbers, according to the method in the present embodiment, in 2.1 chromatographic condition determination samples
In five kinds of chlorogenic acid compositions content, the results are shown in Table 2.
5 kinds of component content measurement results in table 2 Gynura procumbens (Lour.) Merr. extractum
Embodiment 3 one surveys to comment method methodological study more
The calculating of 1 relative correction factor, on the basis of embodiment 2, calculates relative correction factor with multiple mass concentration points
(fk/s), computing formula：fk/s=(Cs×Ak)/(Ck×As), composition quality concentration computing formula to be measured：Ck′=(Cs×Ak′)/
(fk/s×As), wherein CsFor object of reference mass concentration, AsFor object of reference chromatographic peak area, CkFor other matched group mass concentrations, Ak
Compare chromatographic peak peak area, C for otherk′For constituent mass concentration to be measured, Ak′For component chromatographic peak peak area to be measured, result is shown in
Table 3.
The f with chlorogenic acid as object of reference for the table 3k/s(Supplements method)
2 correction factor repeatability are investigated
This effects 2 sets of chromatographic system DIONEX UltiMate 3000 and Agilent1100 of different experiments rooms,
And difference chromatographic column DIONEX C18、WATERS C18To fk/sImpact, its RSD ＜ 3.12%.Show in different experiments room, no
With under chromatographic system and different chromatographic column, there is good repeatability.
3 component chromatographic peak positioning to be measured
3.1 utilize relative retention time (RT) difference qualitative：Know the retention time at internal standard peak, add relative retention time
Difference, judges further according to peak shape, can either correctly judge the accurate location of target peak.Using chlorogenic acid and last peak
4,5-Dicaffeoylquinic acid, two point correction, its correction equation of deriving, with DIONEX C18、WATERS C18Detached chlorogenic acid and different green former
Sour C retention time is independent variable X, and the chlorogenic acid that other chromatographic columns measure and the actual measurement retention time of 4,5-Dicaffeoylquinic acid are dependent variable
Y, its correction equation of can quickly deriving.Again the retention time of detached each composition is brought into equation, you can calculate each composition at it
His instrument and the chromatographic column detached theory corresponding chromatographic peak of appearance time, you can rapid preliminary is set to chromatographic peak.(He Bing, Liu
Gorgeous, Yang Shiyan, a swallow .HPLC mono- survey and comment method to measure 10 kinds of compositions in double green grass or young crops YANHOU PIAN more simultaneously. Chinese herbal medicine, 2013,08 (44):
974-981.)
3.2 1 survey to comment method and external standard method results contrast more：Precision draws 2.2.9 sample in reference substance and embodiment 2 respectively
10 μ L sample introduction under 2.1 chromatographic conditions of embodiment 2 measures, record neochlorogenic acid, chlorogenic acid, 3,5-Dicaffeoylquinic acid, isochlorogenic acid
B, the peak area of 4,5-Dicaffeoylquinic acid, are surveyed using external standard method and one and comment method to calculate its content more.The results are shown in Table 4 it is seen that one surveys comment more
In the multi-target ingredient quality evaluation of Gynura procumbens (Lour.) Merr., application is feasible.
Four components assay result in sample (extractum) in two methods of table 4
Claims (3)
1. a kind of analysis measures Gynura procumbens (Lour.) Merr. Content of Chlorogenic Acid, neochlorogenic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid and 4,5-Dicaffeoylquinic acid
HPLC method it is characterised in that the chromatographic condition of described HPLC method is：
DIONEX UltiMate 3000 high performance liquid chromatograph；DIONEX C18 chromatographic column, 250 × 4.6mm, 5 μm, mobile phase
For acetonitrile -0.1w/v% phosphate aqueous solution, gradient elution：0～65min, 9%～27% acetonitrile；65～70min, 27%～9%
Acetonitrile；Total time is 70min, mobile phase volume flow 1.0mL/min, sample size 10 μ L, Detection wavelength 326nm, 30 DEG C of column temperature.
2. a kind of Gynura procumbens (Lour.) Merr. Content of Chlorogenic Acid, neochlorogenic acid, the external standard method of 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid and 4,5-Dicaffeoylquinic acid
Method for quantitatively determining, comprises the steps：
(1) solution preparation
Prepared by sample solution：Precision weighs extractum 100-250mg, in 10mL volumetric flask, plus 9mL 50v/v% methanol, Yu Chao
In sound instrument, the ultrasonic 15-30min of 20-40KHz, lets cool, plus 50v/v% methanol shakes up to scale, crosses 0.45 μm of filter membrane, obtains final product sample
Solution；
Described extractum preparation method is as follows：Take Gynura procumbens (Lour.) Merr. that stem 0.5 is dried to pulverize, add the 80v/v% ethanol of 5L every time,
Reflux, extract, 1h at 85 DEG C, extracts 3 times, united extraction liquid concentrate drying obtains extractum altogether；
Prepared by mixed reference substance solution：Weigh neochlorogenic acid, chlorogenic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid respectively
Reference substance is placed in volumetric flask, adds 50v/v% methanol to dissolve and be diluted to scale, shakes up, crosses 0.45 μm of microporous filter membrane, obtain final product
Mixed reference substance solution；
(2) making of standard curve：With after 50v/v% methanol gradient dilution mixture reference substance solution described in claim 1
Sample introduction under chromatographic condition in HPLC method, makes neochlorogenic acid, chlorogenic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, different green former respectively
The standard curve of sour C；
(3) by sample solution sample introduction under the chromatographic condition in HPLC method described in claim 1, former according to fresh green respectively
Acid, chlorogenic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, the standard curve of 4,5-Dicaffeoylquinic acid calculate this five kinds of materials in sample solution
Concentration.
3. a survey of a kind of Gynura procumbens (Lour.) Merr. Content of Chlorogenic Acid, neochlorogenic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid and 4,5-Dicaffeoylquinic acid is many
Evaluation quantity measuring method, comprises the steps：
(1) solution preparation
Prepared by sample solution：Precision weighs extractum 100-250mg, in 10mL volumetric flask, plus 9mL 50v/v% methanol, Yu Chao
In sound instrument, the ultrasonic 15-30min of 20-40KHz, lets cool, plus 50v/v% methanol shakes up to scale, crosses 0.45 μm of filter membrane, obtains final product sample
Solution；
Described extractum preparation method is as follows：Take Gynura procumbens (Lour.) Merr. that stem 0.5 is dried to pulverize, add the 80v/v% ethanol of 5L every time,
Reflux, extract, 1h at 85 DEG C, extracts 3 times, united extraction liquid concentrate drying obtains extractum altogether；
Prepared by chlorogenic acid reference substance solution：Weigh chlorogenic acid reference substance to be placed in volumetric flask, add 50v/v% methanol to dissolve and dilute
Release to scale, shake up, cross 0.45 μm of microporous filter membrane, obtain final product chlorogenic acid reference substance solution；
(2) under the chromatographic condition in HPLC method described in claim 1, respectively will be molten to sample solution and chlorogenic acid reference substance
Liquid sample introduction, calculates the concentration of sample solution Content of Chlorogenic Acid according to conventional methods；
With chlorogenic acid for comparison, 4,5-Dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, neochlorogenic acid relative correction factor fk/sRespectively
For 1.21,1.12,1.07,0.92, according to formula Ck′=(Cs×Ak′)/(fk/s×As) calculate determinand in sample solution respectively
Matter 4,5-Dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, the concentration of neochlorogenic acid, wherein CsFor chlorogenic acid concentration, AsFor chlorogenic acid color
Spectral peak area, Ck′For test substance concentration, Ak′For test substance chromatographic peak peak area.
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Effective date of registration: 20230905 Address after: Room 1303, Building 5, Xijia District, No. 182 Minzu Avenue, Hongshan District, Wuhan City, Hubei Province, 430074 Patentee after: Tang Hebin Address before: 410011 502, Building F1, Lemon Lidu, Xinyao Road Crossing, Tianxin District, Changsha, Hunan Province Patentee before: Tan Yulian |
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Effective date of registration: 20240328 Address after: Room 1501, Block B, Nanxian Commercial Plaza, Nanyuan New Village, North Station Community, Minzhi Street, Longhua District, Shenzhen, Guangdong 518000 Patentee after: Shenzhen Renfu Biotechnology Co.,Ltd. Country or region after: China Address before: Room 1303, Building 5, Xijia District, No. 182 Minzu Avenue, Hongshan District, Wuhan City, Hubei Province, 430074 Patentee before: Tang Hebin Country or region before: China |