CN107389845A - 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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- G—PHYSICS
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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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, it is related to a kind of method for building up and its standard finger-print of pumpkin seeds finger-print.It is an object of the invention to provide a kind of method for building up and its standard finger-print of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds.The inventive method is：First, need testing solution is prepared；2nd, finger-print is made, need testing solution is injected into high performance liquid chromatograph；The finger-print of pumpkin seeds of the present invention has 15 common characteristic peaks, and these common characteristic peaks constitute the fingerprint characteristic of pumpkin seeds, can be as the standard finger-print of pumpkin seeds.The present invention have method it is easy, stably, precision height, favorable reproducibility, the characteristics of being easily achieved.The purposes of pumpkin seeds standard finger-print of the present invention refers to pumpkin seeds finger-print as discriminating and quality control of the standard finger-print for the true and false of pumpkin seeds medicinal material, the place of production and quality.The present invention is applied to field of medicaments.
Description
Technical field
The present invention relates to a kind of method for building up and its standard finger-print of pumpkin seeds finger-print.
Background technology
Pumpkin seeds (Semen Moschatae) are Curcurbitaceae cucurbita plants pumpkin Cucurbita moschata Duch.
Seed.Also known as pumpkin benevolence, pumpkin seeds, " golden melon seed ".Contain abundant unrighted acid, sitosterol, phosphatide, monosaccharide, dimension
The nutrient necessary to human body such as raw element, protein, carrotene and zinc, magnesium, iron.It is low close with expeling parasite, reduction
Spend the effect such as lipoprotein cholesterol, anti-inflammatory, anti-oxidant, alleviation hypertension, toxicity very little.Contain a large amount of phosphatide in pumpkin seeds, pass through
Often eating pumpkin seeds can prevent mineral matter from being condensed in the urethral system of people, be allowed to external with urine ejection, can prevent the hair of kidney stone
It is raw.Importantly, active component and abundant Zn-ef ficiency in pumpkin seeds, have health-care effect to prostate, often eat pumpkin
Son, there is good medicinal efficacy to preventing and improving man's prostatic disorders.Pumpkin seeds simultaneously and simply medicine-food two-purpose
Chinese medicine.China's pumpkin planting scope is very wide, and species is various, pumpkin seeds aboundresources, has been acknowledged as special efficacy health food, has had
Very wide exploitation prospect.Because of situations such as the place of production of Chinese medicine, source, collecting season, processing method complexity are, it is necessary to adopt
Its quality is controlled with more scientific, rational method, this is safely and effectively very necessary to ensure clinical application.Finger-print
It has been widely recognized as at present as a kind of technology for carrying out quality control to Chinese medicine on the whole, its advantage is compared with one-component very
It is more obvious to multicomponent qualitative-and-quantitative method.At present in document there is not yet on pumpkin seeds finger-print research report,
Therefore, it is scientificlly and effectively to control its inherent quality, it is necessary to establish the finger-print of pumpkin seeds.
The content of the invention
It is an object of the invention to provide a kind of method for building up and its standard of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds
Finger-print.
The method for building up of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds of the present invention, it is to carry out according to the following steps：
First, the preparation of need testing solution：A, pumpkin seeds medicinal material is taken, the petroleum ether of 10 times of amounts is added after crushing, is flowed back 3 times,
2~3h every time；After residue is dried, the mass percentage concentrations for adding 10 times of amounts are 95% ethanol, are flowed back 2 times, every time 2~
4h, merge each resulting solution, filter, be concentrated under reduced pressure into dry, obtain medicinal extract；
B, it is upper on macroporous resin chromatography after medicinal extract is dissolved with a small amount of distilled water, first with distilling 4 cylinders of water washing
Product, then obtained with 4 column volumes of ethanol elution that mass concentration is 30%, collection eluent, the eluent that is then concentrated under reduced pressure to doing
To drying solid C；
C, the drying solid C for obtaining step b, the solution that concentration is 1~2mg/mL is dissolved into chromatogram methanol, uses micropore
Filter membrane is filtered, and filtrate is need testing solution；
2nd, the making of finger-print：The need testing solution that step 1 is obtained injects high performance liquid chromatograph, with volume ratio
For 1:1 methanol and the mixed solution of acetonitrile are A phases, using volume ratio as 0.2:The phosphoric acid of (100~105) and the mixed liquor of water are
B phases carry out gradient elution；Wherein, high-efficient liquid phase chromatogram condition is：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, the μ L of sample size 10；Analysis supplies on this condition
Test sample solution, obtain the finger-print of pumpkin seeds；
3rd, the determination of standard finger-print：15 common characteristic peaks are determined；Using No. 9 peaks as control peak, common characteristic peaks
Relative retention time be respectively：No. 1 common characteristic peaks relative retention time, which is that 0.462 ± 0.028%, No. 2 peaks are relative, to be retained
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%, No. 7 peak relative retention times are that 0.911 ± 0.004%, No. 8 peak relative retention times are 0.969 ± 0.072%, 9
Number peak relative retention time is that 1.000, No. 10 peak relative retention times are 1.028 ± 0.042%, No. 11 peak relative retention times
For 1.099 ± 0.038%, No. 12 peak relative retention times are that 1.135 ± 0.003%, No. 13 peak relative retention times are 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 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 pumpkin seeds quality control.
Beneficial effects of the present invention：
The pumpkin seeds standard finger-print of the present invention has 15 common characteristic peaks, and these common characteristic peaks constitute pumpkin seeds
Fingerprint characteristic, can be as the standard finger-print of pumpkin seeds.The pumpkin seeds standard finger-print of the present invention is as standard fingerprint
Collection of illustrative plates can be used in pumpkin seeds quality control after more batches of samples are verified, for evaluating and controlling pumpkin seeds medicinal material on the whole
Quality, the one-sidedness that pumpkin seeds total quality is judged because only determining one, two chemical composition was both avoided, had been reduced again for matter
Measure possibility that is up to standard and artificially adding control composition.The method for building up of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds of the present invention
With repeatability is strong, precision is high and reliable and stable, is easy to the advantages of grasp, the true and false, the place of production suitable for pumpkin seeds medicinal material
With the discriminating and quality control of quality.
Brief description of the drawings
Fig. 1 is the pumpkin seeds standard finger-print that experiment 1 provides；Wherein 1 to 15 is 15 common characteristic peaks of pumpkin seeds；
Fig. 2 is that S1-S10 is 10 batches of pumpkin seeds medicinal materials fingerprints.
Embodiment
Technical solution of the present invention is not limited to act embodiment set forth below, in addition between each embodiment
Any combination.
Embodiment one：The method for building up of the efficient liquid-phase chromatograph finger print atlas of present embodiment pumpkin seeds, be by
Following steps are carried out：
First, the preparation of need testing solution：A, pumpkin seeds medicinal material is taken, the petroleum ether of 10 times of amounts is added after crushing, is flowed back 3 times,
2~3h every time；After residue is dried, the mass percentage concentrations for adding 10 times of amounts are 95% ethanol, are flowed back 2 times, every time 2~
4h, merge each resulting solution, filter, be concentrated under reduced pressure into dry, obtain medicinal extract；
B, it is upper on macroporous resin chromatography after medicinal extract is dissolved with a small amount of distilled water, first with distilling 4 cylinders of water washing
Product, then obtained with 4 column volumes of ethanol elution that mass concentration is 30%, collection eluent, the eluent that is then concentrated under reduced pressure to doing
To drying solid C；
C, the drying solid C for obtaining step b, the solution that concentration is 1~2mg/mL is dissolved into chromatogram methanol, uses micropore
Filter membrane is filtered, and filtrate is need testing solution；
2nd, the making of finger-print：The need testing solution that step 1 is obtained injects high performance liquid chromatograph, with volume ratio
For 1:1 methanol and the mixed solution of acetonitrile are A phases, using volume ratio as 0.2:The phosphoric acid of (100~105) and the mixed liquor of water are
B phases carry out gradient elution；Wherein, high-efficient liquid phase chromatogram condition is：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, the μ L of sample size 10；Analysis supplies on this condition
Test sample solution, obtain the finger-print of pumpkin seeds.
Embodiment two：Present embodiment is unlike embodiment one：Miillpore filter in step 1
Filtering is filtered using 0.45 μm of miillpore filter.It is other identical with embodiment one.
Embodiment three：Present embodiment is unlike embodiment one or two：Stone described in step 1
The boiling range of oily ether is 60~90 DEG C.It is other identical with embodiment one or two.
Embodiment four：Unlike one of present embodiment and embodiment one to three：It is terraced in step 2
Spending eluting order is：0min → 22min → 50min → 60min → 75min, volume ratio 1:1 methanol and acetonitrile mixed solution
1% → 15% → 20% → 22% → 25%.It is other identical with one of embodiment one to three.
Embodiment five：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, and these common characteristic peaks constitute south
The fingerprint characteristic of melon seeds, can be as the standard finger-print of pumpkin seeds.
Embodiment six：Present embodiment is unlike embodiment five：The standard fingerprint figure of pumpkin seeds
Spectrum has 15 common characteristic peaks；It is respectively by control peak, the relative retention time of common characteristic peaks of No. 9 peaks：No. 1 common characteristic
Peak relative retention time is that 0.462 ± 0.028%, No. 2 peak relative retention times are the relative guarantor in 0.505 ± 0.006%, No. 3 peaks
It is that 0.707 ± 0.008%, No. 4 peak relative retention times are that 0.775 ± 0.032%, No. 5 peak relative retention times are to stay the time
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 relative
Retention time is 1.028 ± 0.042%, No. 11 peak relative retention times when to be that 1.099 ± 0.038%, No. 12 peaks are relative retain
Between be that 1.135 ± 0.003%, No. 13 peak relative retention times are 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%, and this 15 common characteristic peaks constitute pumpkin
The fingerprint characteristic of son, can be as the standard finger-print of pumpkin seeds.It is other identical with embodiment five.
Embodiment seven：The purposes of present embodiment pumpkin seeds standard finger-print refers to that pumpkin seeds finger-print is made
It is used for for standard finger-print in pumpkin seeds quality control.
The pumpkin seeds standard finger-print of present embodiment can be used for as standard finger-print after more batches of samples are verified
In pumpkin seeds quality control, for evaluating and controlling the quality of pumpkin seeds medicinal material on the whole, both avoided because only determining one, two
Individual chemical composition and judge the one-sidedness of pumpkin seeds total quality, reduce again for requisite quality and artificially add control composition
Possibility.
Beneficial effects of the present invention are verified by tests below：
Experiment 1：
1st, test method：The method for building up of the efficient liquid-phase chromatograph finger print atlas of this experiment pumpkin seeds, it is according to the following steps
Carry out：
First, the preparation of need testing solution：A, pumpkin seeds medicinal material is taken, the petroleum ether (60~90 of 10 times of amounts is added after crushing
DEG C), flow back 3 times, each 2h.After residue is dried, 95% ethanol of 10 times of amounts is added, is flowed back 2 times, each 2h, is merged each
Resulting solution, filtering, is concentrated under reduced pressure into dry, obtains medicinal extract.
B, it is upper on macroporous resin chromatography after medicinal extract is dissolved with a small amount of distilled water, first with distilling 4 cylinders of water washing
Product, then obtained with 4 column volumes of ethanol elution that mass concentration is 30%, collection eluent, the eluent that is then concentrated under reduced pressure to doing
To drying solid C；
C, the drying solid C for obtaining step b, the solution that concentration is 1mg/mL is dissolved into chromatogram methanol, is filtered with micropore
Film is filtered, and filtrate is need testing solution；
2nd, the making of finger-print：The need testing solution that step 1 is obtained injects high performance liquid chromatograph, with volume ratio
For 1:1 methanol and the mixed solution of acetonitrile are A phases, using volume ratio as 0.2:100 phosphoric acid is that B phases are carried out with the mixed liquor of water
Gradient elution；Wherein, high-efficient liquid phase chromatogram condition is：Chromatographic column filler Thermo C18, specification are 250 × 4.6mm, 5 μm；Post
Temperature is 35 DEG C；Flow velocity 0.8mL/min, Detection wavelength 264nm, the μ L of sample size 10；Need testing solution is analyzed on this condition, is obtained
The finger-print of pumpkin seeds；
2nd, test apparatus：HITACHI L-2000 high performance liquid chromatographs；HITACHI L-2400 UV-detectors；AT-
130 chromatographic column column ovens；HITACHI D-2000 chromatographic work stations.
3rd, 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 experiment, 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 Measure software (middle Chinese
Pharmacopoeia commission of people republic, version in 2004), it is determined that 15 common characteristic peaks, using No. 9 peaks as control peak, common characteristic peaks
Relative retention time be respectively：No. 1 common characteristic peaks relative retention time, which is that 0.462 ± 0.028%, No. 2 peaks are relative, to be retained
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%, No. 7 peak relative retention times are that 0.911 ± 0.004%, No. 8 peak relative retention times are 0.969 ± 0.072%, 9
Number peak relative retention time is that 1.000, No. 10 peak relative retention times are 1.028 ± 0.042%, No. 11 peak relative retention times
For 1.099 ± 0.038%, No. 12 peak relative retention times are that 1.135 ± 0.003%, No. 13 peak relative retention times are 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 as the standard finger-print of pumpkin seeds.
The standard finger-print for the pumpkin seeds that this experiment obtains is as shown in Figure 1.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, carried for the quality of overall evaluation pumpkin seeds
Good foundation is supplied.
4th, methodological study
4.1 method reappearance tests
Take with a collection of 6 parts of pumpkin seeds medicinal material, prepare and supply according to the preparation method of pumpkin seeds need testing solution in test method
Test sample solution, determining fingerprint pattern is carried out, the relative retention time and relative peak area that share peak are counted respectively, as a result
As shown in table 1, RSD% is no more than 3%, shows that the method reappearance of the present invention is good.
4.2 finger-print precision tests
Take the need testing solution of method reappearance test a, continuous sample introduction 5 times, respectively to the relative reservation at shared peak
Time and relative peak area are counted, and RSD% is no more than 3% (such as table 2), as a result show that the method precision of the present invention is good
It is good.
4.3 finger-print stability tests
Take the need testing solution of method reappearance test a, carry out determining fingerprint pattern, investigate need testing solution 48
Stability in hour, the relative retention time and relative peak area that share peak are counted respectively, RSD% is no more than
3% (such as table 3), as a result show that need testing solution is stable in 48 hours.
Above result of the test shows that the assay method is reliable and stable, and finger-print is relatively stable.
The reproducible test results of table 1 (n=6)
The Precision test result of table 2 (n=5)
The stability test result (n=5) of table 3
Claims (5)
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
Suddenly：
First, the preparation of need testing solution：A, pumpkin seeds medicinal material is taken, the petroleum ethers of 10 times of amounts are added after crushing, is flowed back 3 times, every time 2
~3h；After residue is dried, the mass percentage concentration for adding 10 times of amounts is 95% ethanol, flows back 2 times, every time 2~4h, merges
Each resulting solution, filtering, is concentrated under reduced pressure into dry, obtains medicinal extract；
B, it is upper on macroporous resin chromatography after medicinal extract is dissolved with a small amount of distilled water, first with distilling 4 column volumes of water washing, then
With 4 column volumes of ethanol elution that mass concentration is 30%, eluent is collected, the eluent that is then concentrated under reduced pressure is done to doing
Dry solid C；
C, the drying solid C for obtaining step b, the solution that concentration is 1~2mg/mL is dissolved into chromatogram methanol, uses miillpore filter
Filtered, filtrate is need testing solution；
2nd, the making of finger-print：The need testing solution that step 1 is obtained injects high performance liquid chromatograph, using volume ratio as 1:
1 methanol and the mixed solution of acetonitrile are A phases, using volume ratio as 0.2:The phosphoric acid of (100~105) and the mixed liquor of water are B phases
Carry out gradient elution；Wherein, high-efficient liquid phase chromatogram condition is：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, the μ L of sample size 10；Need testing solution is analyzed on this condition,
Obtain the finger-print of pumpkin seeds.
2. a kind of method for building up of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds according to claim 1, its 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, its feature exist
The boiling range of the petroleum ether described in step 1 is 60~90 DEG C.
4. a kind of method for building up of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds according to claim 1, its feature exist
Gradient elution order is in step 2：0min → 22min → 50min → 60min → 75min, volume ratio 1:1 methanol and
Acetonitrile mixed solution 1% → 15% → 20% → 22% → 25%.
5. the pumpkin obtained using a kind of method for building up of the efficient liquid-phase chromatograph finger print atlas of pumpkin seeds described in claim 1
Substandard finger-print, it is characterised in that the standard finger-print of pumpkin seeds has 15 common characteristic peaks；Using No. 9 peaks as control
Peak, the relative retention time of common characteristic peaks are respectively：No. 1 common characteristic peaks relative retention time is 0.462 ± 0.028%, 2
Number peak relative retention time is that 0.505 ± 0.006%, No. 3 peak relative retention times are that 0.707 ± 0.008%, No. 4 peaks are relative
Retention time is that 0.775 ± 0.032%, No. 5 peak relative retention times are 0.840 ± 0.013%, No. 6 peak relative retention times
For 0.864 ± 0.046%, No. 7 peak relative retention times be 0.911 ± 0.004%, No. 8 peak relative retention times be 0.969 ±
0.072%, No. 9 peak relative retention times are that 1.000, No. 10 peak relative retention times are 1.028 ± 0.042%, No. 11 peak phases
It is that 1.099 ± 0.038%, No. 12 peak relative retention times are the relative reservation in 1.135 ± 0.003%, No. 13 peaks to retention time
Time is that 1.227 ± 0.029%, No. 14 peak relative retention times are that 1.316 ± 0.023%, No. 15 peak relative retention times are
1.392 ± 0.047%, this 15 common characteristic peaks constitute the fingerprint characteristic of pumpkin seeds.
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