CN107064324B - A kind of identification method and content assaying method of the HPLC finger-print of trigone polyphenol components - Google Patents
A kind of identification method and content assaying method of the HPLC finger-print of trigone polyphenol components Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 39
- 150000008442 polyphenolic compounds Chemical class 0.000 title claims abstract description 29
- 235000013824 polyphenols Nutrition 0.000 title claims abstract description 27
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 69
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 52
- 239000000523 sample Substances 0.000 claims abstract description 29
- 235000019441 ethanol Nutrition 0.000 claims abstract description 23
- 239000012085 test solution Substances 0.000 claims abstract description 22
- 238000010828 elution Methods 0.000 claims abstract description 17
- 239000006071 cream Substances 0.000 claims abstract description 15
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000012488 sample solution Substances 0.000 claims abstract description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- 238000005259 measurement Methods 0.000 claims abstract description 9
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 239000012535 impurity Substances 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 238000010992 reflux Methods 0.000 claims abstract description 8
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 7
- 235000019253 formic acid Nutrition 0.000 claims abstract description 7
- 229920006122 polyamide resin Polymers 0.000 claims abstract description 7
- QAIPRVGONGVQAS-DUXPYHPUSA-N trans-caffeic acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-DUXPYHPUSA-N 0.000 claims description 36
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims description 32
- NGSWKAQJJWESNS-UHFFFAOYSA-N 4-coumaric acid Chemical compound OC(=O)C=CC1=CC=C(O)C=C1 NGSWKAQJJWESNS-UHFFFAOYSA-N 0.000 claims description 30
- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 claims description 19
- 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 claims description 18
- 235000004883 caffeic acid Nutrition 0.000 claims description 18
- 229940074360 caffeic acid Drugs 0.000 claims description 18
- QAIPRVGONGVQAS-UHFFFAOYSA-N cis-caffeic acid Natural products OC(=O)C=CC1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-UHFFFAOYSA-N 0.000 claims description 18
- 239000013558 reference substance Substances 0.000 claims description 18
- 229940090248 4-hydroxybenzoic acid Drugs 0.000 claims description 16
- 239000002253 acid Substances 0.000 claims description 16
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 claims description 15
- NGSWKAQJJWESNS-ZZXKWVIFSA-M 4-Hydroxycinnamate Natural products OC1=CC=C(\C=C\C([O-])=O)C=C1 NGSWKAQJJWESNS-ZZXKWVIFSA-M 0.000 claims description 15
- DFYRUELUNQRZTB-UHFFFAOYSA-N Acetovanillone Natural products COC1=CC(C(C)=O)=CC=C1O DFYRUELUNQRZTB-UHFFFAOYSA-N 0.000 claims description 15
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 claims description 15
- 235000001785 ferulic acid Nutrition 0.000 claims description 15
- 229940114124 ferulic acid Drugs 0.000 claims description 15
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 claims description 15
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 claims description 15
- 238000001914 filtration Methods 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 11
- 239000000047 product Substances 0.000 claims description 10
- 239000011347 resin Substances 0.000 claims description 8
- 229920005989 resin Polymers 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 6
- 238000004090 dissolution Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 2
- 238000002242 deionisation method Methods 0.000 claims 2
- 238000010521 absorption reaction Methods 0.000 abstract description 3
- 230000014759 maintenance of location Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 3
- 244000046052 Phaseolus vulgaris Species 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 239000002304 perfume Substances 0.000 description 3
- 235000013599 spices Nutrition 0.000 description 3
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 239000004952 Polyamide Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 150000007965 phenolic acids Chemical class 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000003252 repetitive effect Effects 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 239000005711 Benzoic acid Substances 0.000 description 1
- 241000233945 Typhaceae Species 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- PJVNDJOBKYOMBU-UHFFFAOYSA-N formic acid;phenol Chemical compound OC=O.OC1=CC=CC=C1 PJVNDJOBKYOMBU-UHFFFAOYSA-N 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- -1 phenolic acid class of p-Coumaric Acid Chemical class 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Cosmetics (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The present invention relates to the identification methods and content assaying method of a kind of HPLC finger-print of trigone polyphenol components, chromatographic condition and system suitability use Kromasil C18 chromatographic column (4.6mm × 250mm, 5 μm), column temperature is 25 DEG C, Detection wavelength is 254nm, and volume flow 0.85mLmin-1, sample volume is 10 μ L, -0.1% formic acid of methanol is mobile phase, gradient elution；The preparation of test solution: ethyl alcohol is heated to reflux, ethyl alcohol is recovered under reduced pressure and is condensed into clear cream, add 4 times of calorimetric water as sample solution, it is added on polyamide resin column and rinses removal of impurities, obtain test solution, close 10 μ 1 of absorption test solution, inject liquid chromatograph, measurement is to get trigone total polyphenols finger-print is obtained.
Description
1, technical field
The invention belongs to pharmaceutical technology fields, are related to a kind of identification method of the HPLC finger-print of trigone polyphenol components
And content assaying method.
2, background technique
Trigone is the dry tuber of Sparganiaceae plant rhizoma scirpi (SparganiumstoleniferumBuch.-Ham),
Belong to dissipating blood stasis with potent drugs class Chinese medicine.It is referred to as " key medicine of Removing Blood Stasis " by modern age name doctor Zhang Xichun trigone.2015 editions " pharmacopeia " trigone items
Under, only routine inspection item (including the identification of microscopical characters, thin layer, moisture, total ash and extract etc.), without index ingredient
Content control method.Control method is simpler, fuzzy, can not truly react the quality of trigone, guarantee clinical application
Validity and safety.It is studied by platelet aggregation-against and platelet aggregation-against, shows that trigone polyphenol components are that trigone is " broken
Blood is by the stasis of blood " the chief active position of effect.
Therefore, the present invention establishes the identification method and assay of a kind of HPLC finger-print of trigone polyphenol components
Method, and it is effectively controlled the quality of medicinal material of trigone through the invention, it ensure that the clinical efficacy of trigone.
3, summary of the invention
To control the quality of genunie medicinal materials trigone more preferably, the present invention has chosen 10 batches, trigone sample, respectively through poly-
Amide resin enriching and purifying obtains trigone active component polyphenols group, carries out finger-print research and measures simultaneously to hydroxyl
The content of benzoic acid, caffeic acid, 5 kinds of vanillic aldehyde, p-Coumaric Acid and ferulic acid ingredients, be trigone medicinal material overall quality control with
The development of genunie medicinal materials provides reference frame.
One of the objects of the present invention is to provide a kind of identification methods of the HPLC finger-print of trigone polyphenol components.
Method is as follows:
Chromatographic condition and system suitability try: Kromasil C18 chromatographic column (4.6mm × 250mm, 5 μm), column temperature 25
DEG C, Detection wavelength 254nm, volume flow 0.85mLmin-1, sample volume are 10 μ L, and -0.1% formic acid of methanol is flowing
Phase, gradient elution: 0~60min, methanol 5%~100%, number of theoretical plate is calculated by forulic acid peak should be not less than 4000；
The preparation of test solution: taking trigone powder (crossing No. four sieves), and about 10g is accurately weighed, sets 250ml tool plug taper
In bottle, 80% ethyl alcohol 100mL is added, is heated to reflux 60min, cooling, filtering takes subsequent filtrate 50ml, and ethyl alcohol and dense is recovered under reduced pressure
Shorten the clear cream of relative density 1.15 (60 DEG C) into, clear cream adds 4 times of calorimetric water to be stirred to dissolve, and standing is let cool, and as sample solution, is taken
Sample solution is added on polyamide resin column (30g, 1.4cm × 10cm), with 0.5mLmin-1After volume flow passes through resin bed, 4
Amount deionized water rinses removal of impurities again, then 4 times of 70% ethanol elutions of amount, collects efflux, and low temperature concentrate drying adds methanol to dissolve
It is settled to 5ml, is filtered to get test solution；
Measuring method: precision draw 10 μ 1 of test solution, inject liquid chromatograph, measurement to get；
Trigone total polyphenols finger-print is obtained, altogether to 10 shared peaks, is compared through HPLC-DAD-MS analysis and reference substance,
Wherein 5 chromatographic peaks respectively correspond P-hydroxybenzoic acid peak, caffeic acid peak, vanillic aldehyde peak, p-Coumaric Acid peak, forulic acid peak, wash
The de- time is respectively 22.5min, 24.7min, 26.3min, 29.1min, 30.5min.
The second object of the present invention is to provide a kind of content assaying method of trigone polyphenol components
Method is as follows:
Chromatographic condition and system suitability try: Kromasil C18 chromatographic column (4.6mm × 250mm, 5 μm), column temperature 25
DEG C, Detection wavelength 254nm, volume flow 0.85mLmin-1, sample volume are 10 μ L, and -0.1% formic acid of methanol is flowing
Phase, gradient elution: 0~60min, methanol 5%~100%, number of theoretical plate is calculated by forulic acid peak should be not less than 4000；
The preparation of reference substance solution: P-hydroxybenzoic acid, caffeic acid, vanillic aldehyde, p-Coumaric Acid and ferulic acid pair are taken respectively
It is appropriate according to product, it is accurately weighed, add methanol that mixing of every 1mL containing 40.98,36.00,69.78,48.67,16.56 μ g is made and compares
Product solution to get；
The preparation of test solution: taking trigone powder (crossing No. four sieves), and about 10g is accurately weighed, sets 250ml tool plug taper
In bottle, 80% ethyl alcohol 100mL is added, is heated to reflux 60min, cooling, filtering takes subsequent filtrate 50ml, and ethyl alcohol and dense is recovered under reduced pressure
Shorten the clear cream of relative density 1.15 (60 DEG C) into, clear cream adds 4 times of calorimetric water to be stirred to dissolve, and standing is let cool, and as sample solution, is taken
Sample solution is added on polyamide resin column (30g, 1.4cm × 10cm), with 0.5mLmin-1After volume flow passes through resin bed, 4
Amount deionized water rinses removal of impurities again, then 4 times of 70% ethanol elutions of amount, collects efflux, and low temperature concentrate drying adds methanol to dissolve
Be settled to 5ml, filtering to get；
Measuring method: accurate absorption reference substance solution and each 10 μ 1 of test solution respectively, injection liquid chromatograph, measurement,
To obtain the final product；
This product by dry product calculate, containing polyphenol components with P-hydroxybenzoic acid peak, caffeic acid peak, vanillic aldehyde peak, to perfume (or spice)
The total amount meter at beans acid peak, ferulic acid must not be less than 0.03%.
This method not only establishes trigone polyphenol components finger-print under same chromatographic condition, but also determines 5 kinds simultaneously
The content of phenolic acid components, method is easy, has qualitative and quantitative double action.Finger-print the result shows that medicinal material phenolic acid class
Ingredient has good homogeneity.
The method specificity for the trigone polyphenol components finger-print that this method is established is strong, within the scope of Detection wavelength, sample
Product contain much information, and response is higher, and each chromatographic peak peak shape is symmetrical after gradient elution, and separation is preferable.
The P-hydroxybenzoic acid that is measured in this method, caffeic acid, vanillic aldehyde, 5 kinds of phenolic acid class of p-Coumaric Acid and ferulic acid at
Point, wherein ferulic acid has platelet aggregation-against, anti-inflammatory, antioxidation, identical as trigone effect；Caffeic acid, p-Coumaric Acid
With anti-oxidant and effect of scavenging radical, P-hydroxybenzoic acid, vanillic aldehyde are can to destroy the cell of microorganism with antibiotic property
Film makes cell protein denaturation, and inhibits the respiratory enzyme of microbial cell and the activity of electron transmission enzyme system.
4, Detailed description of the invention
Fig. 1: trigone polyphenols compares dactylogram；
A. reference substance chromatogram is mixed；
B. reference fingerprint；
1. P-hydroxybenzoic acid；
2. caffeic acid；
3. vanillic aldehyde；
4. p-Coumaric Acid；
5. ferulic acid
Fig. 2: trigone polyphenols HPLC dactylogram
5, specific embodiment
Form by the following examples is described in further detail above content of the invention again, but should not be by this
The range for being interpreted as the above-mentioned theme of the present invention is only limitted to example below, and all technologies realized based on above content of the present invention are equal
Belong to the scope of the present invention.
The preparation method of 1 trigone polyphenol components of embodiment
Trigone powder (crossing No. four sieves) is taken, about 10g is accurately weighed, sets in 250ml stuffed conical flask, and 80% ethyl alcohol is added
100mL is heated to reflux 60min, cooling, and filtering takes subsequent filtrate 50ml, ethyl alcohol is recovered under reduced pressure and is condensed into relative density 1.15
The clear cream of (60 DEG C)；Clear cream adds 4 times of calorimetric water to be stirred to dissolve, and standing is let cool, as sample solution；Sample solution is taken to be added on polyamide
On resin column (30g, 1.4cm × 10cm), with 0.5mLmin-1After volume flow passes through resin bed, 4 times of amount deionized waters are rinsed
Removal of impurities, then 4 times of 70% ethanol elutions of amount, collect efflux, and low temperature is concentrated and dried, and add methanol dissolution to be settled to 5ml, filter,
To obtain the final product.
The identification method 2 of the HPLC finger-print of 2 trigone polyphenol components of embodiment
The preparation of test solution takes trigone powder (crossing No. four sieves), and about 10g is accurately weighed, sets 250ml tool plug taper
In bottle, 80% ethyl alcohol 100mL is added, is heated to reflux 60min, cooling, filtering takes subsequent filtrate 50ml, and ethyl alcohol and dense is recovered under reduced pressure
Shorten the clear cream of relative density 1.15 (60 DEG C) into；Clear cream adds 4 times of calorimetric water to be stirred to dissolve, and standing is let cool, as sample solution；It takes
Sample solution is added on polyamide resin column (30g, 1.4cm × 10cm), with 0.5mLmin-1After volume flow passes through resin bed, 4
Amount deionized water rinses removal of impurities again, then 4 times of 70% ethanol elutions of amount, collects efflux, and low temperature concentrate drying adds methanol to dissolve
Be settled to 5ml, filtering to get.
Chromatographic condition Kromasil C18 chromatographic column (4.6mm × 250mm, 5 μm)；Column temperature is 25 DEG C；Detection wavelength is
254nm；Volume flow is 0.85mLmin-1；Sample volume is 10 μ L；- 0.1% formic acid of methanol be mobile phase, gradient elution: 0~
60min, methanol 5%~100%.
Precision test takes same test solution, by chromatographic condition, repeats sample introduction 6 times, chromatogram is recorded, with asafoetide
For acid to calculate the relative retention time and relative peak area at each shared peak in finger-print referring to peak, as a result each shared peak is opposite
The RSD of the retention time equal < 1.9% of equal < 0.1%, the RSD of relative peak area, shows that this method precision is preferable.
Stability test takes same test solution, by chromatographic condition, measures respectively in 0,4,8,12,16,18h sample introduction,
Chromatogram is recorded, is to calculate each shared peak relative retention time and relative peak area in finger-print, knot referring to peak with ferulic acid
The RSD equal < 2.3% of equal < 0.2%, the RSD of relative peak area of the relative retention time at each shared peak of fruit, shows that test sample is molten
Liquid is placed 18h at room temperature and is stablized.
Repetitive test takes 6 parts of same batch trigone, prepares test solution by the preparation method of test solution, presses
Chromatographic condition sample introduction records chromatogram, with ferulic acid be referring to peak, calculate in finger-print each shared peak relative retention time and
Relative peak area, the RSD of the relative retention time at the as a result each shared peak equal < 2.0% of equal < 0.3%, the RSD of relative peak area,
Show that this method has preferable repeatability.
The generation of trigone total polyphenols finger-print measures the finger-print of 10 batches of trigones according to the above method, using national medicine
" the traditional Chinese medicine fingerprint similarity evaluation system " 2012.1 editions that the allusion quotation committee provides, establishes the control fingerprint image of trigone total polyphenols
Spectrum, as a result the trigone of all batches can detect 10 shared peaks, be compared with reference substance, determine that No. 1 peak is para hydroxybenzene
Formic acid, No. 2 peaks are caffeic acid, and No. 3 peaks are vanillic aldehyde, and No. 4 peaks are p-Coumaric Acid, and No. 5 peaks are ferulic acid, and elution time is respectively
22.5min,24.7min,26.3min,29.1min,30.5min.Mixing reference substance chromatogram and reference fingerprint are shown in that figure is attached
Fig. 1.
The measurement of trigone sample fingerprint similarity is with the trigone polyphenols group reference fingerprint of above-mentioned generation
As control, " similarity evaluation " 2012.1 editions provided using Chinese Pharmacopoeia Commission is soft
Part calculates the similarity of each chromatogram, and 10 batches of similarities between sample and reference fingerprint are followed successively by 0.845,1,
0.927,0.821,0.958,0.843,0.814,0.907,0.870,0.933, show the finger-prints of 10 batches of samples with compare
Map has higher similarity, sees attached drawing 2.
The content assaying method of 3 trigone polyphenol components of embodiment
The preparation of reference substance solution respectively precision weigh P-hydroxybenzoic acid, caffeic acid, vanillic aldehyde, p-Coumaric Acid and Ah
Wei's acid reference substance is appropriate, add methanol be made mixed reference substance solution to get.
The preparation trigone sample of test solution smashes it through No. 4 sieves in right amount, takes about 10g, accurately weighed, sets 250ml tool
It fills in conical flask, 80% ethyl alcohol 100mL is added, is heated to reflux 60min, cooling, filtering takes subsequent filtrate 50ml, second is recovered under reduced pressure
Alcohol and the clear cream for being condensed into relative density 1.15 (60 DEG C)；Clear cream adds 4 times of calorimetric water to be stirred to dissolve, and standing is let cool, as upper
Sample liquid；Sample solution is taken to be added on polyamide resin column (30g, 1.4cm × 10cm), with 0.5mLmin-1Volume flow passes through tree
After rouge bed, 4 times of amount deionized waters rinse removal of impurities, then 4 times of 70% ethanol elutions of amount, collect efflux, and low temperature is concentrated and dried, adds
Methanol dissolution is settled to 5ml, filtering to get.
Chromatographic condition Kromasil C18 chromatographic column (4.6mm × 250mm, 5 μm)；Column temperature is 25 DEG C；Detection wavelength is
254nm；Volume flow is 0.85mLmin-1；Sample volume is 10 μ L；Number of theoretical plate should be not less than 6000 based on forulic acid peak,
- 0.1% formic acid of methanol is mobile phase, gradient elution: 0~60min, methanol 5%~100%.
Linear relationship investigates accurate absorption 1,2,5,10,15,20,25 μ L of reference substance solution, successively sample introduction, with sample volume
(ng) be abscissa, peak area is ordinate, carry out linear regression, acquire P-hydroxybenzoic acid, caffeic acid, vanillic aldehyde, to perfume (or spice)
The regression equation of beans acid and ferulic acid is shown in Table 1, the results showed that P-hydroxybenzoic acid, caffeic acid, vanillic aldehyde, p-Coumaric Acid and asafoetide
Acid linear relationship within the scope of respective concentration is good.
5 kinds of components regression equations and the range of linearity in 1 trigone total polyphenols of table
Precision test takes same reference substance product solution, by chromatographic condition, repeats sample introduction 6 times, measurement, para hydroxybenzene first
Acid, caffeic acid, vanillic aldehyde, p-Coumaric Acid and forulic acid peak area RSD be respectively 0.4%, 1.8%, 1.4%, 0.9% He
1.1%, show that instrument precision is preferable.
Stability test takes same reference substance solution, by chromatographic condition, measures respectively in 0,4,8,12,16,18h sample introduction,
As a result the RSD of P-hydroxybenzoic acid, caffeic acid, vanillic aldehyde, p-Coumaric Acid and forulic acid peak area is respectively 1.0%, 1.7%,
1.5%, 0.8% and 2.0%, show that test solution is placed 18h at room temperature and stablized.
Repetitive test takes with a collection of 6 parts of trigone sample (S2), test solution is prepared according to the above method, by above-mentioned color
Spectral condition sample introduction measures, as a result the average mass fraction of P-hydroxybenzoic acid, caffeic acid, vanillic aldehyde, p-Coumaric Acid and ferulic acid
Respectively 0.056,0.036,0.121,0.059 and 0.089mgg-1, RSD are respectively 2.2%, 1.7%, 2.1%, 1.1%
With 1.6%, show that this method has preferable repeatability.
Sample recovery rate tests trigone sample (S2) 10g for taking known content, and precision addition reference substance solution is appropriate, presses
The above method prepares test solution, measures by above-mentioned chromatographic condition, the results are shown in Table 2.
The sample recovery rate (n=6) of 5 kinds of effective component in 2 trigone total polyphenols of table
Sample size measurement takes 10 batches of different batches trigone samples respectively, measures according to the method described above, as a result trigone sample
The content range of middle P-hydroxybenzoic acid is 0.045~0.065mgg-1, caffeinic content range be 0.031~
0.045mg·g-1, the content range of vanillic aldehyde is 0.107~0.133mgg-1, the content range of p-Coumaric Acid is 0.050~
0.068mg·g-1, the content range of ferulic acid is 0.075~0.099mgg-1, it is shown in Table 3.
Measurement result (the mgg of 3 10 batches, table, 5 kinds of trigone sample ingredient-1)
This product by dry product calculate, containing polyphenol components with P-hydroxybenzoic acid peak, caffeic acid peak, vanillic aldehyde peak, to perfume (or spice)
The total amount meter at beans acid peak, ferulic acid must not be less than 0.03%.
Claims (2)
1. a kind of identification method of the HPLC finger-print of trigone polyphenol components, it is characterized in that using following methods
Chromatographic condition and system suitability: 4.6mm × 250mm, 5 μm of Kromasil C18 chromatographic columns, column temperature are 25 DEG C,
Detection wavelength is 254nm, and volume flow 0.85mLmin-1, sample volume is 10 μ L, and -0.1% formic acid of methanol is mobile phase, ladder
Degree elution: 0~60min, methanol 5%~100%, number of theoretical plate is calculated by forulic acid peak should be not less than 4000；
The preparation of test solution: taking trigone powder to cross No. four sieves, and about 10g is accurately weighed, sets in 250ml stuffed conical flask, adds
Enter 80% ethyl alcohol 100mL, be heated to reflux 60min, cooling, filtering takes subsequent filtrate 50ml, ethyl alcohol is recovered under reduced pressure and is condensed into 60 DEG C
The clear cream of lower relative density 1.15, clear cream add 4 times of calorimetric water to be stirred to dissolve, and standing is let cool, and as sample solution, sample solution is taken to add
In on 30g, 1.4cm × 10cm polyamide resin column, with 0.5mLmin-1After volume flow passes through resin bed, 4 times of amount deionizations
Water rinses removal of impurities, then 4 times of 70% ethanol elutions of amount, collects efflux, and low temperature is concentrated and dried, and methanol dissolution is added to be settled to 5ml,
Filtering is to get test solution；
Measuring method: precision draw 10 μ 1 of test solution, inject liquid chromatograph, measurement to get；
Trigone total polyphenols finger-print is obtained, altogether to 10 shared peaks, is compared through HPLC-DAD-MS analysis and reference substance, wherein 5
A chromatographic peak respectively corresponds P-hydroxybenzoic acid peak, caffeic acid peak, vanillic aldehyde peak, p-Coumaric Acid peak, forulic acid peak, elution time
Respectively 22.5min, 24.7min, 26.3min, 29.1min, 30.5min.
2. a kind of content assaying method of trigone polyphenol components, it is characterized in that using following methods,
Chromatographic condition and system suitability: 4.6mm × 250mm, 5 μm of Kromasil C18 chromatographic columns, column temperature are 25 DEG C,
Detection wavelength is 254nm, and volume flow 0.85mLmin-1, sample volume is 10 μ L, and -0.1% formic acid of methanol is mobile phase, ladder
Degree elution: 0~60min, methanol 5%~100%, number of theoretical plate is calculated by forulic acid peak should be not less than 4000；
The preparation of reference substance solution: P-hydroxybenzoic acid, caffeic acid, vanillic aldehyde, p-Coumaric Acid and ferulic acid reference substance are taken respectively
In right amount, accurately weighed, add methanol that mixing reference substance of every 1mL containing 40.98,36.00,69.78,48.67,16.56 μ g is made molten
Liquid to get；
The preparation of test solution: taking trigone powder to cross No. four sieves, and about 10g is accurately weighed, sets in 250ml stuffed conical flask, adds
Enter 80% ethyl alcohol 100mL, be heated to reflux 60min, cooling, filtering takes subsequent filtrate 50ml, ethyl alcohol is recovered under reduced pressure and is condensed into 60 DEG C
The clear cream of lower relative density 1.15, clear cream add 4 times of calorimetric water to be stirred to dissolve, and standing is let cool, and as sample solution, sample solution is taken to add
In on 30g, 1.4cm × 10cm polyamide resin column, with 0.5mLmin-1After volume flow passes through resin bed, 4 times of amount deionizations
Water rinses removal of impurities, then 4 times of 70% ethanol elutions of amount, collects efflux, and low temperature is concentrated and dried, and methanol dissolution is added to be settled to 5ml,
Filtering to get；
Measuring method: it is accurate respectively to draw reference substance solution and each 10 μ 1 of test solution, inject liquid chromatograph, measurement to get；
This product is calculated by dry product, containing polyphenol components with P-hydroxybenzoic acid peak, caffeic acid peak, vanillic aldehyde peak, p-Coumaric Acid
The total amount meter at peak, ferulic acid must not be less than 0.03%.
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