CN107389845B - A kind of method for building up and its standard finger-print of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds - Google Patents
A kind of method for building up and its standard finger-print of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds Download PDFInfo
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- G—PHYSICS
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- 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
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Abstract
A kind of method for building up and its standard finger-print of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds, are related to the method for building up and its standard finger-print of a kind of pumpkin seeds finger-print.The object of the present invention is to provide a kind of method for building up of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds and its standard finger-prints.The method of the present invention are as follows: one, prepare test solution；Two, finger-print is made, test solution is injected into high performance liquid chromatograph；The finger-print of pumpkin seeds of the present invention has 15 common characteristic peaks, these common characteristic peaks constitute the fingerprint characteristic of pumpkin seeds, can be used as the standard finger-print of pumpkin seeds.The present invention has the characteristics that method is easy, stablizes, precision height, favorable reproducibility, is easily achieved.The purposes of pumpkin seeds standard finger-print of the present invention refers to pumpkin seeds finger-print as standard finger-print for the true and false of pumpkin seeds medicinal material, the identification in the place of production and quality and quality control.The present invention is applied to field of medicaments.
Description
Technical field
The present invention relates to the method for building up and its standard finger-print of a kind of pumpkin seeds finger-print.
Background technique
Pumpkin seeds (Semen Moschatae) are Curcurbitaceae cucurbita plants pumpkin Cucurbita moschata Duch.
Seed.Also known as pumpkin benevolence, pumpkin seeds, " golden melon seed ".Unsaturated fatty acid rich in, sitosterol, phosphatide, monosaccharide, dimension
Nutrient necessary to the human bodies such as raw element, protein, carrotene and zinc, magnesium, iron.It is low with expeling helminth, reduction
Density lipoprotein-cholesterol, it is anti-inflammatory, anti-oxidant, alleviate hypertension the effects of, toxicity very little.Contain a large amount of phosphatide in pumpkin seeds,
Often eating pumpkin seeds can prevent minerals from condensing in the urethral system of people, be allowed to be excreted with urine in vitro, can prevent kidney stone
Occur.Importantly, active constituent and Zn-ef ficiency abundant in pumpkin seeds, have health-care effect to prostate, often eat south
Melon seeds have good medicinal efficacy to preventing and improving man's prostatic disorders.Pumpkin seeds are also dual-purpose of drug and food simply simultaneously
Chinese medicine.China's pumpkin planting range is very wide, and many kinds of, pumpkin seeds are resourceful, has been acknowledged as special efficacy health food, tool
There is very wide exploitation prospect.Since the place of production of Chinese medicine, source, collecting season, processing method are complicated, need
Its quality is controlled using more scientific, reasonable method, this is safely and effectively very necessary to guarantee clinical application.Fingerprint image
It composes and is widely recognized as at present as a kind of technology for carrying out quality control to Chinese medicine on the whole, advantage is compared with one-component
Even multicomponent qualitative-and-quantitative method is more obvious.At present there is not yet research report about pumpkin seeds finger-print in document
Road, therefore, scientificlly and effectively to control its inherent quality, it is necessary to establish the finger-print of pumpkin seeds.
Summary of the invention
The object of the present invention is to provide a kind of method for building up of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds and its standards
Finger-print.
The method for building up of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds of the present invention is to sequentially include the following steps:
One, the preparation of test solution: a, taking pumpkin seeds medicinal material, and the petroleum ether of 10 times of amounts is added after crushing, flows back 3 times,
2~3h every time；After residue is dried, the ethyl alcohol that the mass percentage concentration of 10 times of amounts is 95% is added, flows back 2 times, every time 2~
4h merges each acquired solution, and filtering is concentrated to dryness, obtains medicinal extract；
B, on macroporous resin chromatography on after dissolving medicinal extract with a small amount of distilled water, 4 cylinders are first washed with distilled water
Product, then 4 column volumes of ethanol elution for being 30% with mass concentration, collect eluent, and eluent is then concentrated under reduced pressure to doing, obtains
To drying solid C；
C, the drying solid C for obtaining step b is dissolved into the solution that concentration is 1~2mg/mL with chromatography methanol, uses micropore
Filter membrane is filtered, and filtrate is test solution；
Two, the production of finger-print: the test solution that step 1 is obtained injects high performance liquid chromatograph, with volume ratio
Be A phase for the methanol of 1:1 and the mixed solution of acetonitrile, be 0.2:(100~105 with volume ratio) phosphoric acid and the mixed liquor of water be
B phase carries out gradient elution；Wherein, high-efficient liquid phase chromatogram condition are as follows: chromatographic column filler Thermo C18, specification be 250 ×
4.6mm, 5 μm；Column temperature is 35 DEG C；Flow velocity 0.8mL/min, Detection wavelength 264nm, 10 μ L of sample volume；Analysis supplies with this condition
Test sample solution obtains the finger-print of pumpkin seeds；
Three, 15 common characteristic peaks the determination of standard finger-print: have been determined；It is control peak, common characteristic peaks with No. 9 peaks
Relative retention time be respectively as follows: No. 1 common characteristic peaks relative retention time to be that 0.462 ± 0.028%, No. 2 peaks are opposite retain
Time is that 0.505 ± 0.006%, No. 3 peak relative retention times are that 0.707 ± 0.008%, No. 4 peak relative retention times are
0.775 ± 0.032%, No. 5 peak relative retention times be 0.840 ± 0.013%, No. 6 peak relative retention times be 0.864 ±
0.046%, it is 0.969 ± 0.072%, 9 that No. 7 peak relative retention times, which are 0.911 ± 0.004%, No. 8 peak relative retention times,
It is 1.028 ± 0.042%, No. 11 peak relative retention times that number peak relative retention time, which is 1.000, No. 10 peak relative retention times,
It is 1.099 ± 0.038%, No. 12 peak relative retention times be 1.135 ± 0.003%, No. 13 peak relative retention times is 1.227
± 0.029%, No. 14 peak relative retention times be 1.316 ± 0.023%, No. 15 peak relative retention times be 1.392 ±
0.047%, this 15 common characteristic peaks constitute the fingerprint characteristic of pumpkin seeds, can be used as the standard finger-print of pumpkin seeds.
Further, the boiling range of petroleum ether described in step 1 is 60~90 DEG C.
The purposes of pumpkin seeds standard finger-print of the present invention refers to that pumpkin seeds finger-print is used for as standard finger-print
In the control of pumpkin protonatomic mass.
Beneficial effects of the present invention:
Pumpkin seeds standard finger-print of the invention has 15 common characteristic peaks, these common characteristic peaks constitute pumpkin seeds
Fingerprint characteristic, can be used as the standard finger-print of pumpkin seeds.Pumpkin seeds standard finger-print of the invention is as standard fingerprint
Map can be used for after more batches of samples are verified in the control of pumpkin protonatomic mass, for evaluating and controlling pumpkin seeds medicinal material on the whole
Quality had not only avoided and has determined the one-sidedness of pumpkin seeds total quality because only measuring one, two chemical component, but also reduced for matter
It measures up to standard and artificially adds a possibility that compareing ingredient.The method for building up of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds of the present invention
Have the advantages that repeatability is strong, precision is high and reliable and stable, is easy to grasp, the true and false, the place of production suitable for pumpkin seeds medicinal material
Identification and quality control with quality.
Detailed description of the invention
Fig. 1 is to test the 1 pumpkin seeds standard finger-print provided；Wherein 1 to 15 be pumpkin seeds 15 common characteristic peaks；
Fig. 2 is that S1-S10 is 10 batches of pumpkin seeds medicinal materials fingerprints.
Specific embodiment
The technical solution of the present invention is not limited to the following list, further includes between each specific embodiment
Any combination.
Specific embodiment 1: the method for building up of the efficient liquid-phase chromatograph finger print atlas of present embodiment pumpkin seeds, be by
Following steps carry out:
One, the preparation of test solution: a, taking pumpkin seeds medicinal material, and the petroleum ether of 10 times of amounts is added after crushing, flows back 3 times,
2~3h every time；After residue is dried, the ethyl alcohol that the mass percentage concentration of 10 times of amounts is 95% is added, flows back 2 times, every time 2~
4h merges each acquired solution, and filtering is concentrated to dryness, obtains medicinal extract；
B, on macroporous resin chromatography on after dissolving medicinal extract with a small amount of distilled water, 4 cylinders are first washed with distilled water
Product, then 4 column volumes of ethanol elution for being 30% with mass concentration, collect eluent, and eluent is then concentrated under reduced pressure to doing, obtains
To drying solid C；
C, the drying solid C for obtaining step b is dissolved into the solution that concentration is 1~2mg/mL with chromatography methanol, uses micropore
Filter membrane is filtered, and filtrate is test solution；
Two, the production of finger-print: the test solution that step 1 is obtained injects high performance liquid chromatograph, with volume ratio
Be A phase for the methanol of 1:1 and the mixed solution of acetonitrile, be 0.2:(100~105 with volume ratio) phosphoric acid and the mixed liquor of water be
B phase carries out gradient elution；Wherein, high-efficient liquid phase chromatogram condition are as follows: chromatographic column filler Thermo C18, specification be 250 ×
4.6mm, 5 μm；Column temperature is 35 DEG C；Flow velocity 0.8mL/min, Detection wavelength 264nm, 10 μ L of sample volume；Analysis supplies with this condition
Test sample solution obtains the finger-print of pumpkin seeds.
Specific embodiment 2: the present embodiment is different from the first embodiment in that: the miillpore filter in step 1
Filtering is filtered using 0.45 μm of miillpore filter.It is other same as the specific embodiment one.
Specific embodiment 3: the present embodiment is different from the first and the second embodiment in that: stone described in step 1
The boiling range of oily ether is 60~90 DEG C.It is other the same as one or two specific embodiments.
Specific embodiment 4: unlike one of present embodiment and specific embodiment one to three: terraced in step 2
Spend eluting order are as follows: 0min → 22min → 50min → 60min → 75min, volume ratio are the methanol and acetonitrile mixed solution of 1:1
1% → 15% → 20% → 22% → 25%.It is other identical as one of specific embodiment one to three.
Specific embodiment 5: present embodiment uses a kind of method for building up of pumpkin seeds efficient liquid-phase chromatograph finger print atlas
Obtained pumpkin seeds standard finger-print.
The pumpkin seeds standard finger-print of present embodiment has 15 common characteristic peaks, these common characteristic peaks constitute south
The fingerprint characteristic of melon seeds can be used as the standard finger-print of pumpkin seeds.
Specific embodiment 6: present embodiment is unlike specific embodiment five: the standard fingerprint figure of pumpkin seeds
Spectrum has 15 common characteristic peaks；It is control peak with No. 9 peaks, the relative retention time of common characteristic peaks is respectively as follows: No. 1 common characteristic
Peak relative retention time is that 0.462 ± 0.028%, No. 2 peak relative retention times are the opposite guarantor in 0.505 ± 0.006%, No. 3 peaks
It is that 0.775 ± 0.032%, No. 5 peak relative retention times are that stay the time, which be 0.707 ± 0.008%, No. 4 peak relative retention times,
0.840 ± 0.013%, No. 6 peak relative retention times be 0.864 ± 0.046%, No. 7 peak relative retention times be 0.911 ±
0.004%, No. 8 peak relative retention times are that 0.969 ± 0.072%, No. 9 peak relative retention times are that 1.000, No. 10 peaks are opposite
Retention time is that 1.028 ± 0.042%, No. 11 peak relative retention times are 1.099 ± 0.038%, No. 12 peaks with respect to when retaining
Between be 1.135 ± 0.003%, No. 13 peak relative retention times be that 1.227 ± 0.029%, No. 14 peak relative retention times are
1.316 ± 0.023%, No. 15 peak relative retention times are 1.392 ± 0.047%, this 15 common characteristic peaks constitute pumpkin
The fingerprint characteristic of son, can be used as the standard finger-print of pumpkin seeds.It is other identical as specific embodiment five.
Specific embodiment 7: the purposes of present embodiment pumpkin seeds standard finger-print refers to that pumpkin seeds finger-print is made
It is standard finger-print in the control of pumpkin protonatomic mass.
The pumpkin seeds standard finger-print of present embodiment can be used for after more batches of samples are verified as standard finger-print
In the control of pumpkin protonatomic mass, for evaluating and controlling the quality of pumpkin seeds medicinal material on the whole, both avoided because of only measurement one, two
A chemical component and the one-sidedness for determining pumpkin seeds total quality, and reduce and artificially add control ingredient for requisite quality
Possibility.
Beneficial effects of the present invention are verified by following tests:
Test 1:
1, test method: the method for building up of the efficient liquid-phase chromatograph finger print atlas of this test pumpkin seeds is according to the following steps
It carries out:
One, the preparation of test solution: a, taking pumpkin seeds medicinal material, and the petroleum ether (60~90 of 10 times of amounts is added after crushing
DEG C), it flows back 3 times, each 2h.After residue is dried, 95% ethyl alcohol of 10 times of amounts is added, flows back 2 times, each 2h, merges each
Acquired solution, filtering, is concentrated to dryness, obtains medicinal extract.
B, on macroporous resin chromatography on after dissolving medicinal extract with a small amount of distilled water, 4 cylinders are first washed with distilled water
Product, then 4 column volumes of ethanol elution for being 30% with mass concentration, collect eluent, and eluent is then concentrated under reduced pressure to doing, obtains
To drying solid C；
C, the drying solid C for obtaining step b is dissolved into the solution that concentration is 1mg/mL with chromatography methanol, is filtered with micropore
Film is filtered, and filtrate is test solution；
Two, the production of finger-print: the test solution that step 1 is obtained injects high performance liquid chromatograph, with volume ratio
It is A phase for the methanol of 1:1 and the mixed solution of acetonitrile, for the phosphoric acid of 0.2:100 is that B phase carries out with the mixed liquor of water with volume ratio
Gradient elution；Wherein, high-efficient liquid phase chromatogram condition are as follows: chromatographic column filler Thermo C18, specification be 250 × 4.6mm, 5 μm；Column
Temperature is 35 DEG C；Flow velocity 0.8mL/min, Detection wavelength 264nm, 10 μ L of sample volume；Test solution is analyzed with this condition, is obtained
The finger-print of pumpkin seeds；
2, test apparatus: HITACHI L-2000 high performance liquid chromatograph；HITACHI L-2400 UV detector；AT-
130 chromatographic column column ovens；HITACHI D-2000 chromatographic work station.
3, test material: 10 batches of pumpkin seeds medicinal materials are from Heilungkiang, Hebei, Xinjiang, Gansu, the Inner Mongol, Shandong and other places.
According to the method for building up of the efficient liquid-phase chromatograph finger print atlas of the pumpkin seeds of this test, 10 batches of pumpkin seeds medicinal materials are built
Efficient liquid-phase chromatograph finger print atlas is found, obtained spectrogram imports chromatographic fingerprints of Chinese materia medica similarity calculation software (middle Chinese
Pharmacopoeia commission, people republic, version in 2004), it is determined that 15 common characteristic peaks are control peak, common characteristic peaks with No. 9 peaks
Relative retention time be respectively as follows: No. 1 common characteristic peaks relative retention time to be that 0.462 ± 0.028%, No. 2 peaks are opposite retain
Time is that 0.505 ± 0.006%, No. 3 peak relative retention times are that 0.707 ± 0.008%, No. 4 peak relative retention times are
0.775 ± 0.032%, No. 5 peak relative retention times be 0.840 ± 0.013%, No. 6 peak relative retention times be 0.864 ±
0.046%, it is 0.969 ± 0.072%, 9 that No. 7 peak relative retention times, which are 0.911 ± 0.004%, No. 8 peak relative retention times,
It is 1.028 ± 0.042%, No. 11 peak relative retention times that number peak relative retention time, which is 1.000, No. 10 peak relative retention times,
It is 1.099 ± 0.038%, No. 12 peak relative retention times be 1.135 ± 0.003%, No. 13 peak relative retention times is 1.227
± 0.029%, No. 14 peak relative retention times be 1.316 ± 0.023%, No. 15 peak relative retention times be 1.392 ±
0.047%, this 15 common characteristic peaks constitute the fingerprint characteristic of pumpkin seeds, can be used as the standard finger-print of pumpkin seeds.
The standard finger-print for the pumpkin seeds that this test obtains is as shown in Figure 1.It as seen from Figure 1, can by this method
Clearly to obtain the standard finger-print of the pumpkin seeds with 15 common characteristic peaks, mentioned for the quality of overall evaluation pumpkin seeds
Good foundation is supplied.
4, methodological study
4.1 method reappearance tests
It takes with a collection of 6 parts of medicinal material of pumpkin seeds, prepares and supply according to the preparation method of pumpkin seeds test solution in test method
Test sample solution carries out determining fingerprint pattern, counts respectively to the relative retention time and relative peak area at shared peak, as a result
As shown in table 1, RSD% is no more than 3%, shows that method reproducibility of the invention is good.
4.2 finger-print precision tests
It takes the test solution of method reappearance test a, continuous sample introduction 5 times, the opposite of shared peak is retained respectively
Time and relative peak area are counted, and RSD% is no more than 3% (such as table 2), and method precision of the invention is good as the result is shown
It is good.
4.3 finger-print stability tests
It takes the test solution of method reappearance test a, carries out determining fingerprint pattern, investigate test solution 48
Stability in hour, respectively counts the relative retention time and relative peak area at shared peak, RSD% is no more than
3% (such as table 3), test solution is stable within 48 hours as the result is shown.
The above test result shows that the measuring method is reliable and stable, and finger-print is relatively stable.
1 reproducible test results of table (n=6)
2 Precision test result of table (n=5)
3 stability test result (n=5) of table
Claims (3)
1. a kind of method for building up of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds, it is characterised in that this method includes following step
It is rapid:
One, the preparation of test solution: a, taking pumpkin seeds medicinal material, and the petroleum ethers of 10 times of amounts are added after crushing, flows back 3 times, and every time 2
~3h；After residue is dried, the ethyl alcohol that the mass percentage concentration of 10 times of amounts is 95% is added, flows back 2 times, every time 2~4h, merges
Each acquired solution, filtering, is concentrated to dryness, obtains medicinal extract；
B, on macroporous resin chromatography on after dissolving medicinal extract with a small amount of distilled water, 4 column volumes are first washed with distilled water, then
4 column volumes of ethanol elution for being 30% with mass concentration collect eluent, and eluent is then concentrated under reduced pressure to doing, is done
Dry solid C；
C, the drying solid C for obtaining step b is dissolved into the solution that concentration is 1~2mg/mL with chromatography methanol, uses miillpore filter
It is filtered, filtrate is test solution；
Two, the production of finger-print: the test solution that step 1 is obtained injects high performance liquid chromatograph, with volume ratio for 1:
The mixed solution of 1 methanol and acetonitrile is A phase, is 0.2:(100~105 with volume ratio) phosphoric acid and the mixed liquor of water be B phase
Carry out gradient elution；Wherein, high-efficient liquid phase chromatogram condition are as follows: chromatographic column filler Thermo C18, specification are 250 × 4.6mm, 5 μ
m；Column temperature is 35 DEG C；Flow velocity 0.8mL/min, Detection wavelength 264nm, 10 μ L of sample volume；Test solution is analyzed with this condition,
Obtain the finger-print of pumpkin seeds；
Gradient elution sequence in step 2 are as follows: 0min → 22min → 50min → 60min → 75min, volume ratio are the methanol of 1:1
With acetonitrile mixed solution 1% → 15% → 20% → 22% → 25%.
2. a kind of method for building up of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds according to claim 1, feature exist
Filtering with microporous membrane in step 1 is filtered using 0.45 μm of miillpore filter.
3. a kind of method for building up of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds according to claim 1, feature exist
The boiling range of the petroleum ether described in step 1 is 60~90 DEG C.
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