CN105203670B - Establishing method of HPLC (high performance liquid chromatography) fingerprints of loofah sponge - Google Patents
Establishing method of HPLC (high performance liquid chromatography) fingerprints of loofah sponge Download PDFInfo
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- CN105203670B CN105203670B CN201510732396.3A CN201510732396A CN105203670B CN 105203670 B CN105203670 B CN 105203670B CN 201510732396 A CN201510732396 A CN 201510732396A CN 105203670 B CN105203670 B CN 105203670B
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- fingerprints
- retinervus luffae
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- luffae fructuss
- loofah sponge
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Abstract
The invention discloses an establishing method of HPLC (high performance liquid chromatography) fingerprints of loofah sponge and relates to an establishing method of the fingerprints of the loofah sponge, standard fingerprints established with the method and an application. The method comprises steps as follows: step one, preparation of a solution for test products; step two, preparation of the fingerprints: the solution for test products is injected to a high-performance liquid chromatograph. The fingerprints of the loofah sponge have seven common characteristic peaks, the common characteristic peaks form the fingerprint feature of the loofah sponge, and the fingerprints of the loofah sponge can serve as the standard fingerprints of the loofah sponge. The method has the characteristics of simplicity, convenience, stability, high precision, good reproducibility and easiness in mastering. The fingerprints of the loofah sponge can be applied to authenticity, production place and quality identification and quality control of loofah sponge medicinal materials as the standard fingerprints. The invention applies to the field of medicine.
Description
Technical field
The present invention relates to a kind of method for building up of Retinervus Luffae Fructuss finger printing and its standard finger-print and purposes.
Background technology
Retinervus Luffae Fructuss, i.e. the dimension pipe of the dry mature fruit of cucurbitaceous plant Fructus Luffae Luffa cylindrica (L.) Roem.
Beam.The compositions such as sterol, saponin, flavone, phenols, protein, aminoacid and organic acid are mainly contained, with wind-dispelling, dredging collateral, work
Effect of blood, stimulating milk secretion, for arthralgia pain contracture, distending pain in the chest and hypochondrium, galactostasiss, acute mastitis such as swell and ache the disease.Modern study shows, Retinervus Luffae Fructuss
Effect with Ischemic myocardium, and at aspects such as blood fat reducing, suppression increased weight, antiinflammatory, antalgic and sedative and antiallergic
Also achieve good effect.In terms of quality control, 2010 editions《Pharmacopoeia of People's Republic of China》One has been recorded Retinervus Luffae Fructuss
Medical material, but only carried out powder discriminating.As the place of production of Chinese medicine, source, collecting season are complicated, need to adopt more section
Learn, rational method controls its quality, this is safely and effectively very necessary to ensure clinical application.Finger printing as from
A kind of technology for carrying out quality control on the whole to Chinese medicine is widely recognized as at present, and its advantage is even multigroup compared with one-component
Qualitative-and-quantitative method is divided to become apparent from.There is not yet the research with regard to Retinervus Luffae Fructuss finger printing is reported in current document, therefore, it is
Its inherent quality is controlled scientificlly and effectively, it is necessary to set up the finger printing of Retinervus Luffae Fructuss.
The content of the invention
It is an object of the invention to provide a kind of method for building up of the efficient liquid-phase chromatograph finger print atlas of Retinervus Luffae Fructuss and its standard
Finger printing and purposes.
The method for building up of the efficient liquid-phase chromatograph finger print atlas of Retinervus Luffae Fructuss of the present invention, is to carry out according to the following steps：
First, the preparation of need testing solution：A, Retinervus Luffae Fructuss medical material is taken, plus the mass percentage concentration of 10 times of quality is 75% second
Alcohol, soaks 12h, ultrasonic 30min, filters, and collects solid formation A and liquid phase thing A respectively, solid formation A is added the matter of 10 times of quality
Amount percentage concentration is filtered for soaking 8h, ultrasonic 30min in 75% ethanol, collects liquid phase thing B, is then mixed liquid phase thing A and B,
Lixiviating solution is obtained, lixiviating solution concentrating under reduced pressure is obtained into extractum；
B, by extractum with after a small amount of distillation water dissolution in polyamide chromatographic column, first with 4 column volumes of distilled water wash,
Again with 4 column volumes of the ethanol elution that mass concentration is 30%, eluent is collected, then concentrating under reduced pressure eluent is obtained to dry
Drying solid C；
C, the drying solid C for obtaining step b, are dissolved into the solution that concentration is 2mg/mL with methanol, are entered with microporous filter membrane
Row is filtered, and filtrate is need testing solution；
2nd, the making of finger printing：The need testing solution injection high performance liquid chromatograph that step one is obtained, with volume ratio
For 1:1 methanol and the mixed solution of acetonitrile are A phases, and mass percentage concentration is that 0.5% acetic acid aqueous solution carries out gradient for B phases
Eluting；Wherein, high-efficient liquid phase chromatogram condition is：Chromatographic column filler Thermo C18, specification be 250 × 4.6mm, 5 μm；Column temperature is
35℃；Flow velocity 0.8mL/min, Detection wavelength 280nm, 20 μ L of sample size；Need testing solution is analyzed with this understanding, obtains Fructus Luffae
The finger printing of network.
3rd, the determination of standard finger-print：Determine 7 common characteristic peaks；With No. 7 peaks as compareing peak, common characteristic peaks
Relative retention time be respectively：No. 1 common characteristic peaks relative retention time is the relative reservation in 0.368 ± 0.003%, No. 2 peaks
Time for 0.433 ± 0.01%, No. 3 peak relative retention time for 0.462 ± 0.025%, No. 4 peak relative retention time is
0.613 ± 0.016%, No. 5 peak relative retention time be 0.667 ± 0.02%, No. 6 peak relative retention time be 0.881 ±
0.028%, No. 7 peak relative retention time are 1.000, and above-mentioned common characteristic peaks constitute the fingerprint characteristic of Retinervus Luffae Fructuss, can conduct
The standard finger-print of Retinervus Luffae Fructuss.
The purposes of Retinervus Luffae Fructuss standard finger-print of the present invention refers to that Retinervus Luffae Fructuss finger printing is used for as standard finger-print
In Retinervus Luffae Fructuss quality control.
Beneficial effects of the present invention：
The Retinervus Luffae Fructuss standard finger-print of the present invention has 7 common characteristic peaks, and these common characteristic peaks constitute Retinervus Luffae Fructuss
Fingerprint characteristic, can be used as the standard finger-print of Retinervus Luffae Fructuss.The Retinervus Luffae Fructuss standard finger-print of the present invention is used as standard fingerprint
Collection of illustrative plates can be used in Retinervus Luffae Fructuss quality control Jing after many batches of sample checkings, for evaluating on the whole and controlling Retinervus Luffae Fructuss medical material
Quality, had both avoided and had judged the one-sidedness of Retinervus Luffae Fructuss total quality because only determining one, two chemical compositions, reduced as matter again
Measure probability 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 Retinervus Luffae Fructuss of the present invention
Have the advantages that repeatability is strong, precision is high and reliable and stable, be easy to grasp, it is adaptable to the true and false of Retinervus Luffae Fructuss medical material, the place of production
Discriminating and quality control with quality.
Description of the drawings
Fig. 1 is to test the 1 Retinervus Luffae Fructuss standard finger-print for providing；Wherein 1 to 7 is 7 common characteristic peaks of Retinervus Luffae Fructuss；
Fig. 2 is 10 batches of Retinervus Luffae Fructuss medicinal materials fingerprints for S1-S10.
Specific embodiment
Technical solution of the present invention is not limited to act specific embodiment set forth below, also including between each specific embodiment
Combination in any.
Specific embodiment one：The method for building up of the efficient liquid-phase chromatograph finger print atlas of present embodiment Retinervus Luffae Fructuss, be by
Following steps are carried out：
First, the preparation of need testing solution：A, Retinervus Luffae Fructuss medical material is taken, plus the mass percentage concentration of 10 times of quality is 75% second
Alcohol, soaks 12h, ultrasonic 30min, filters, and collects solid formation A and liquid phase thing A respectively, solid formation A is added the matter of 10 times of quality
Amount percentage concentration is filtered for soaking 8h, ultrasonic 30min in 75% ethanol, collects liquid phase thing B, is then mixed liquid phase thing A and B,
Lixiviating solution is obtained, lixiviating solution concentrating under reduced pressure is obtained into extractum；
B, by extractum with after a small amount of distillation water dissolution in polyamide chromatographic column, first with 4 column volumes of distilled water wash,
Again with 4 column volumes of the ethanol elution that mass concentration is 30%, eluent is collected, then concentrating under reduced pressure eluent is obtained to dry
Drying solid C；
C, the drying solid C for obtaining step b, are dissolved into the solution that concentration is 2mg/mL with methanol, are entered with microporous filter membrane
Row is filtered, and filtrate is need testing solution；
2nd, the making of finger printing：The need testing solution injection high performance liquid chromatograph that step one is obtained, with volume ratio
For 1:1 methanol and the mixed solution of acetonitrile are A phases, and mass percentage concentration is that 0.5% acetic acid aqueous solution carries out gradient for B phases
Eluting；Wherein, high-efficient liquid phase chromatogram condition is：Chromatographic column filler Thermo C18, specification be 250 × 4.6mm, 5 μm；Column temperature is
35℃；Flow velocity 0.8mL/min, Detection wavelength 280nm, 20 μ L of sample size；Need testing solution is analyzed with this understanding, obtains Fructus Luffae
The finger printing of network.
Specific embodiment two：Present embodiment from unlike specific embodiment one：Microporous filter membrane in step one
Filtration is filtered using 0.45 μm of microporous filter membrane.Other are identical with specific embodiment one.
Specific embodiment three：Present embodiment from unlike specific embodiment one or two：In step 2, gradient is washed
De- order is：0min → 20min → 50min → 80min, volume ratio are 1:1 methanol and acetonitrile mixed solution 5% → 16% →
25% → 40%.Other are identical with specific embodiment one or two.
Specific embodiment four：Present embodiment adopts a kind of method for building up of Retinervus Luffae Fructuss efficient liquid-phase chromatograph finger print atlas
The Retinervus Luffae Fructuss standard finger-print for obtaining.
The Retinervus Luffae Fructuss standard finger-print of present embodiment has 7 common characteristic peaks, and these common characteristic peaks constitute silk
The fingerprint characteristic of melon network, can be used as the standard finger-print of Retinervus Luffae Fructuss.
Specific embodiment five：Present embodiment from unlike specific embodiment four：The standard fingerprint figure of Retinervus Luffae Fructuss
Spectrum has 7 common characteristic peaks；It is respectively with No. 7 peaks as compareing peak, the relative retention time of common characteristic peaks：No. 1 common characteristic
It is the relative reservation in 0.433 ± 0.01%, No. 3 peaks that peak relative retention time is 0.368 ± 0.003%, No. 2 peak relative retention time
Time for 0.462 ± 0.025%, No. 4 peak relative retention time for 0.613 ± 0.016%, No. 5 peak relative retention time is
0.667 ± 0.02%, No. 6 peak relative retention time are that 0.881 ± 0.028%, No. 7 peak relative retention time are 1.000, and this 7
Individual common characteristic peaks constitute the fingerprint characteristic of Retinervus Luffae Fructuss, can be used as the standard finger-print of Retinervus Luffae Fructuss.Other be embodied as
Mode four is identical.
Specific embodiment six：The purposes of present embodiment Retinervus Luffae Fructuss standard finger-print refers to that Retinervus Luffae Fructuss finger printing is made
It is used in Retinervus Luffae Fructuss quality control for standard finger-print.
The Retinervus Luffae Fructuss standard finger-print of present embodiment can be used for Jing after many batches of sample checkings as standard finger-print
In Retinervus Luffae Fructuss quality control, for evaluating and controlling the quality of Retinervus Luffae Fructuss medical material on the whole, both avoided because only determining one, two
Individual chemical composition and judge the one-sidedness of Retinervus Luffae Fructuss total quality, reduce as requisite quality again and artificially add control composition
Probability.
Beneficial effects of the present invention are verified by tests below：
Test 1：
1st, test method：The method for building up of the efficient liquid-phase chromatograph finger print atlas of this test Retinervus Luffae Fructuss, is according to the following steps
Carry out：
First, the preparation of need testing solution：A, Retinervus Luffae Fructuss medical material is taken, plus the mass percentage concentration of 10 times of quality is 75% second
Alcohol, soaks 12h, ultrasonic 30min, filters, and collects solid formation A and liquid phase thing A respectively, solid formation A is added the matter of 10 times of quality
Amount percentage concentration is filtered for soaking 8h, ultrasonic 30min in 75% ethanol, collects liquid phase thing B, is then mixed liquid phase thing A and B,
Lixiviating solution is obtained, lixiviating solution concentrating under reduced pressure is obtained into extractum；
B, by extractum with after a small amount of distillation water dissolution in polyamide chromatographic column, first with 4 column volumes of distilled water wash,
Again with 4 column volumes of the ethanol elution that mass concentration is 30%, eluent is collected, then concentrating under reduced pressure eluent is obtained to dry
Drying solid C；
C, the drying solid C for obtaining step b, are dissolved into the solution that concentration is 2mg/mL with methanol, are entered with microporous filter membrane
Row is filtered, and filtrate is need testing solution；
2nd, the making of finger printing：The need testing solution injection high performance liquid chromatograph that step one is obtained, with volume ratio
For 1:1 methanol and the mixed solution of acetonitrile are A phases, and mass percentage concentration is that 0.5% acetic acid aqueous solution carries out gradient for B phases
Eluting；Wherein, high-efficient liquid phase chromatogram condition is：1st, chromatographic column filler Thermo C18, specification be 250 × 4.6mm, 5 μm；Column temperature
For 35 DEG C；Using gradient elution mode 0min → 20min → 50min → 80min, volume ratio is 1:1 methanol and acetonitrile mixing
Solution 5% → 16% → 25% → 40%；Flow velocity 0.8mL/min, Detection wavelength 280nm, 20 μ L of sample size；
2nd, test apparatuses：HITACHI L-2000 high performance liquid chromatographs；HITACHI L-2400 UV-detector；AT-
130 chromatographic column column ovens；HITACHI D-2000 chromatographic work stations.
3rd, test material：10 batches of Retinervus Luffae Fructuss medical materials are from Hunan, Shandong, Anhui, Zhejiang, Guangxi, Shanxi, Jiangsu and other places.
According to the method for building up of the efficient liquid-phase chromatograph finger print atlas of this Retinervus Luffae Fructus tested, 10 batches of Retinervus Luffae Fructuss medical materials are built
Efficient liquid-phase chromatograph finger print atlas are found, the spectrogram for obtaining imports chromatographic fingerprints of Chinese materia medica Similarity Measure software (middle Chinese
Committee of pharmacopeia of people republic, version in 2004), it is determined that 7 common characteristic peaks, with No. 7 peaks as compareing peak, common characteristic peaks
Relative retention time is respectively：When No. 1 common characteristic peaks relative retention time is that 0.368 ± 0.003%, No. 2 peaks retain relatively
Between for 0.433 ± 0.01%, No. 3 peak relative retention time be 0.462 ± 0.025%, No. 4 peak relative retention time be 0.613
± 0.016%, No. 5 peak relative retention time are that 0.667 ± 0.02%, No. 6 peak relative retention time are 0.881 ± 0.028%,
No. 7 peak relative retention time are 1.000, and above-mentioned common characteristic peaks constitute the fingerprint characteristic of Retinervus Luffae Fructuss, can be used as Retinervus Luffae Fructuss
Standard finger-print.
The standard finger-print of the Retinervus Luffae Fructuss that this test is obtained is as shown in Figure 1.As seen from Figure 1, can by this method
Clearly to obtain the standard finger-print of the Retinervus Luffae Fructuss with 7 common characteristic peaks, it is that the quality of overall evaluation Retinervus Luffae Fructuss is carried
Good foundation is supplied.
4th, methodological study
4.1 method reappearance tests
Take with 6 parts of a collection of Retinervus Luffae Fructuss medical material, prepare according to the preparation method of Retinervus Luffae Fructuss need testing solution in test method and supply
Test sample solution, carries out determining fingerprint pattern, and the relative retention time and relative peak area to having peak is counted respectively, as a result
As shown in table 1, RSD% is less than 3%, shows that method of the present invention repeatability is good.
4.2 finger printing precision tests
The need testing solution for taking method reappearance test is a, continuous sample introduction 5 times, respectively the relative reservation to total peak
Time and relative peak area are counted, and RSD% is less than 3% (such as table 2), as a result shows that method of the present invention precision is good
It is good.
4.3 finger printing stability tests
The need testing solution for taking method reappearance test is a, carries out determining fingerprint pattern, investigates need testing solution 48
Stability in hour, is counted to the relative retention time and relative peak area at total peak respectively, and RSD% is less than
3% (such as table 3), as a result shows 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 printing 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 (2)
1. a kind of method for building up of the efficient liquid-phase chromatograph finger print atlas of Retinervus Luffae Fructuss, it is characterised in that the method is entered according to the following steps
OK：
First, the preparation of need testing solution：A, Retinervus Luffae Fructuss medical material is taken, plus the ethanol that the mass percentage concentration of 10 times of quality is 75%,
Immersion 12h, ultrasonic 30min, filter, and collect solid formation A and liquid phase thing A respectively, solid formation A is added the quality hundred of 10 times of quality
Point concentration be 75% ethanol in soak 8h, ultrasonic 30min, filter, collect liquid phase thing B, then liquid phase thing A and B are mixed, is obtained
To lixiviating solution, lixiviating solution concentrating under reduced pressure is obtained into extractum；
B, by extractum with distillation water dissolution after on in polyamide chromatographic column, first with 4 column volumes of distilled water wash, then use quality
Concentration is 30% 4 column volumes of ethanol elution, collects eluent, and then concentrating under reduced pressure eluent obtains drying solid to dry
C；
C, the drying solid C for obtaining step b, are dissolved into the solution that concentration is 2mg/mL with methanol, were carried out with microporous filter membrane
Filter, filtrate are need testing solution；
2nd, the making of finger printing：The need testing solution injection high performance liquid chromatograph that step one is obtained, with volume ratio as 1:
1 methanol and the mixed solution of acetonitrile are A phases, and mass percentage concentration is that 0.5% acetic acid aqueous solution carries out gradient for B phases and washes
It is de-；Wherein, high-efficient liquid phase chromatogram condition is：Chromatographic column filler Thermo C18, specification be 250 × 4.6mm, 5 μm；Column temperature is 35
℃；Flow velocity 0.8mL/min, Detection wavelength 280nm, 20 μ L of sample size；Need testing solution is analyzed with this understanding, obtains Retinervus Luffae Fructuss
Finger printing；
Wherein in step 2, gradient elution order is：0min → 20min → 50min → 80min, volume ratio are 1:1 methanol and
Acetonitrile mixed solution 5% → 16% → 25% → 40%.
2. the method for building up of the efficient liquid-phase chromatograph finger print atlas of a kind of Retinervus Luffae Fructuss according to claim 1, its feature exist
Filtering with microporous membrane in step one is filtered using 0.45 μm of microporous filter membrane.
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| CN102814168A (en) * | 2012-09-18 | 2012-12-12 | 济南大学 | Preparation method and application of loofah sponge surface malachite green molecular imprinting adsorbing material |
| CN104004833A (en) * | 2014-05-09 | 2014-08-27 | 广东省农业科学院蔬菜研究所 | EST-SSR core primer group developed on basis of transcriptome sequence of towel gourd and application thereof |
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