CN105158369A - Establishment method of HPLC (High Performance Liquid Chromatography) fingerprint spectrum of allinase inactivated extract of six-peal red garlics - Google Patents
Establishment method of HPLC (High Performance Liquid Chromatography) fingerprint spectrum of allinase inactivated extract of six-peal red garlics Download PDFInfo
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Abstract
The invention discloses an establishment method of an HPLC (High Performance Liquid Chromatography) fingerprint spectrum of an allinase inactivated extract of six-peal red garlics. The establishment method comprises the following specific steps of preparing six-peal red bulbs which belong to a peculiar variety in Baodi County into a test product solution by a solid-phase extraction step and the like; performing HPLC detection under certain conditions to obtain the HPLC fingerprint spectrum of the allinase inactivated extract of the six-peal red garlics; analyzing the HPLC fingerprint spectrums of ten batches of samples to determine a common characteristic peak and obtaining a standard fingerprint spectrum which can provide a reliable basis for the identification of varieties and production places of the six-peal red garlics and control over the production quality. The establishment method disclosed by the invention has the advantages of convenience in operation, high stability, high specificity, good reproducibility, more characteristic peaks and high analytic efficiency; by using the establishment method disclosed by the invention, the authenticity and production place of the six-peal red garlics can be identified, and the internal quality of the six-peal red garlics can be evaluated comprehensively, accurately and effectively.
Description
The present invention obtains " Tianjin Urban Committee on Agriculture main project (201302030) and Tianjin Normal University's research for application and development fund " subsidizes.
Technical field
The invention belongs to natural plants analysis technical field, relate to the construction method of natural plants finger-print, especially the construction method of Baodi " six lobes are red " Garlic enzyme-deactivating extract high performance liquid chromatography (HPLC) finger-print and standard finger-print thereof.
Background technology
Garlic (
alliumsativumL.) Liliaceae allium, biennial herb plant, food medicine dual-purpose, is worldwide widely cultivated.At present, China is maximum garlic producing country, and 69.2 ten thousand hectares, domestic plantation garlic, accounts for about 1/2 of world's cultivated area, and the total production of China garlic accounts for Gross World Product more than 3/4, output 1208.8 ten thousand tons.Garlic is as requisite vegetables and flavouring in people's life, nutritious, containing the multiple chemical composition useful to human body, comprise wherein sulfur-bearing volatile matter 43 kinds, sulfuration sulfinic acid (as allicin) ester class 13 kinds, 9 kinds, amino acid, peptide class 8 kinds, glucosides class 12 kinds, enzyme 11 kinds etc.The equal edible of garlic bulb, seedling and scape.Garlic also has stronger medical value, has the effects such as sterilization, anti-inflammatory, antiviral, anticancer, reducing blood lipid.
The peculiar kind of Baodi " six lobes are red " garlic, the fine and close consolidation of the head of garlic, generally about every 6 lobes, single column encloses seat, without adding wedge, without polyphyll, garlic clove is pure white tender and crisp, pungent thick-flavor, easily preserves, resistance to transport, disease resistance is comparatively strong, and pungent is soft agreeable to the taste, and raw, prepared food is used all good." six lobes are red " garlic, with its excellent economical character, abundant nutritional labeling and good mouthfeel, remains the status of famous-brand and high-quality special product always, deeply likes by domestic and international consumer, has found a good sale in multiple countries and regions such as Japan, Korea S and Southeast Asia at present.But because qualitative control is backward in technique, cause kind to occur the sign of degenerating, become the bottleneck of this famous-brand and high-quality kind of restriction scientific, large-scale development further.
Fingerprint pattern technology has become one of most effective means of internationally recognized control Chinese medicine or natural drug quality.It can reflect the distribution of Multiple components in Chinese medicine or its preparation more comprehensively, accurately, intuitively, be a kind of comprehensively, macroscopic view with quantifiable discrimination method.Use fingerprint pattern technology, by the identification at principal character peak in finger-print or the mensuration of ratio, the true and false of Chinese crude drug and Chinese patent drug can be differentiated, the homogeneity of effective evaluation product quality and stability.
Allinnase is the class endogenous enzymes in garlic, and be present in Cell vacuoles, alliin is then present in tenuigenin with stable form.When garlic bulb receives External Force Acting, eucaryotic cell structure is destroyed, and allinnase contacts with alliin, enzyme digestion reaction occurs, generates a series of alliin metabolic product as diallyl disulphide and Diallytrisin etc.As can be seen here, in leaching process, make allinase deactivation, fully can react the chemical composition in garlic under native state.But, up to now, about the allinase deactivation extract finger-print of the peculiar garlic cultivar of Baodi " six lobes are red " have not been reported.
In view of above deficiency, be necessary that invention one sets up the new method of Baodi " six lobes are red " Garlic enzyme-deactivating extract finger-print, enable easy, stable, can repeat, comprehensively, the accurate quality evaluating " six lobes are red " garlic, thus the distinguishing ability both strengthened " six lobes are red " the garlic true and false and the place of production, comprehensive assessment and the control of " six lobes are red " garlic quality can be improved again, compensate for the deficiency of existing detection method, make it science, perfect more.
Summary of the invention
The technical problem to be solved in the present invention is the deficiency for existing Baodi " six lobes are red " Garlic quality management detection method, and provides a kind of method for building up of " six lobes are red " Garlic enzyme-deactivating extractive HPLC fingerprint.The method is by being prepared into allinase deactivation extract solution to be measured by garlic, be separated concentrated through solid phase extraction techniques, detect via high performance liquid chromatograph analysis again, obtain finger-print, for the True-false distinguish of " six lobes are red " garlic and quality control provide reliable basis and technical method.
The method for building up of Baodi of the present invention " six lobes are red " Garlic enzyme-deactivating extractive HPLC fingerprint, is characterized in that: the method is undertaken by following step:
(1) preparation of Garlic enzyme-deactivating extract solution to be measured: take Baodi " six lobes are red " garlic bulb 20g, add 25-30ml distilled water, 90 DEG C of enzyme 1h that go out, crushing grinding, add methanol constant volume to 50ml, the centrifugal 15min of 5000 × g, get supernatant through 0.45 μm of filtering with microporous membrane;
(2) preparation of Garlic enzyme-deactivating extract need testing solution: C18 solid phase extraction column, through 5ml methyl alcohol, the activation of 10ml ultrapure water, balances 15min; Solution to be measured crosses post with 4ml/min speed, then uses the drip washing of 5ml ultrapure water, and low vacuum is drained; Again by after 5ml methanol-eluted fractions, under room temperature, low flow velocity nitrogen is concentrated into 0.5-0.8ml, then with methanol constant volume to 1ml, to be measured through 0.45 μm of filtering with microporous membrane;
(3) efficient liquid phase chromatographic analysis: chromatographic column is: EclipsePlusC18,5 μm, 4.6x250mm; Guard column is: EclipsePlusC18Grd, 5 μm, 4.6x12.5mm; Column temperature 35 DEG C; Mobile phase is methyl alcohol and 0.2% aqueous formic acid; Adopt gradient elution mode 0 → 2min → 10min → 20min, methyl alcohol: 60% → 60% → 80% → 80%; Diode array detector determined wavelength 240nm; Sample size 50 μ L; Analyze need testing solution under these conditions, obtain Garlic enzyme-deactivating extractive HPLC fingerprint;
" six lobes are red " Garlic enzyme-deactivating extractive HPLC standard finger-print that the present invention also provides above-mentioned Garlic enzyme-deactivating extractive HPLC fingerprint to obtain.
10 batches of " six lobes are red " garlic samples are prepared into need testing solution as stated above, be separated by said method and detect, use " similarity evaluation 2004 editions " software analysis of Chinese Pharmacopoeia Commission, obtain " six lobes are red " Garlic enzyme-deactivating extract standard finger-print.The method determines 15 common characteristic peaks, its retention time is respectively: 2.16min, 2.748min, 2.94min, 3.379min, 3.813min, 4.719min, 5.839min, 6.439min, 7.074min, 8.002min, 9.131min, 10.615min, 13.069min, 15.314min, 18.325min, relative standard deviation (RSD) <1%, these common characteristic peaks constitute Baodi " six lobes are red " Garlic enzyme-deactivating extractive HPLC standard finger-print.
The method for building up of Baodi disclosed by the invention " six lobes are red " Garlic enzyme-deactivating extractive HPLC fingerprint compared with prior art had crucial part is:
(1) sample pre-treatments is easy, is easy to operation, and adopt solid phase extraction techniques to carry out pre-separation to sample and concentrate, the need testing solution impurities obtained is few, does not pollute chromatographic column, and From Spectral Signal of checking colors interference is little;
(2) chromatographic condition is simple and easy to do, favorable reproducibility;
(3) the present invention can extract the organic components under garlic bulb native state to the full extent, and the HPLC finger-print set up on this basis controls to have more specific aim and practicality to " six lobes are red " Garlic quality.
(4) under chromatographic of the present invention, each chromatographic peak is separated better, can identify the feature of " six lobes are red " garlic chemical composition on the whole.
(5) analysis efficiency of the present invention is high, good stability, high specificity, favorable reproducibility, and characteristic peak is more, utilizes the present invention can carry out the true and false and place of production discriminating to Baodi " six lobes are red " garlic, and can evaluate its interior quality quality comprehensively, exactly.
Accompanying drawing illustrates:
Fig. 1 is " six lobes are red " Garlic enzyme-deactivating extractive HPLC fingerprint;
Fig. 2 is " six lobes are red " Garlic enzyme-deactivating extractive HPLC standard finger-print.
Embodiment
Below in conjunction with embodiment, the present invention is described, the scheme of embodiment described here, do not limit the present invention, one of skill in the art can make improvements and change according to spirit of the present invention, these described improvement and change all should be considered as within the scope of the invention, and scope of the present invention and essence are limited by claim.Various raw material used all has commercially available.
Embodiment 1:
The method for building up of Baodi " six lobes are red " Garlic enzyme-deactivating extractive HPLC fingerprint, the method step is as follows:
(1) sample, kit instrument: Baodi " six lobes are red " garlic, use Agilent 1200 type high performance liquid chromatograph, reagent is chromatographically pure or analyzes pure;
(2) preparation of Garlic enzyme-deactivating extract solution to be measured: take Baodi " six lobes are red " garlic bulb 20g, add 25-30ml distilled water, 90 DEG C of enzyme 1h that go out, crushing grinding, add methanol constant volume to 50ml, the centrifugal 15min of 5000 × g, get supernatant through 0.45 μm of filtering with microporous membrane;
(3) preparation of Garlic enzyme-deactivating extract need testing solution: C18 solid phase extraction column, through 5ml methyl alcohol, the activation of 10ml ultrapure water, balances 15min; Solution to be measured crosses post with 4ml/min speed, then uses the drip washing of 5ml ultrapure water, and low vacuum is drained; Again by after 5ml methanol-eluted fractions, under room temperature, low flow velocity nitrogen is concentrated into 0.5-0.8ml, then with methanol constant volume to 1ml, to be measured through 0.45 μm of filtering with microporous membrane;
(4) efficient liquid phase chromatographic analysis: chromatographic column is: EclipsePlusC18,5 μm, 4.6x250mm; Guard column is: EclipsePlusC18Grd, 5 μm, 4.6x12.5mm; Column temperature 35 DEG C; Mobile phase is methyl alcohol and 0.2% aqueous formic acid; Adopt gradient elution mode 0 → 2min → 10min → 20min, methyl alcohol: 60% → 60% → 80% → 80%; Diode array detector determined wavelength 240nm; Sample size 50 μ L.
The each test sample liquor of accurate absorption respectively, is separated through high performance liquid chromatograph and detects, obtain Garlic enzyme-deactivating extractive HPLC fingerprint, see Fig. 1 under above-mentioned chromatographic condition.
Embodiment 2:
The method for building up of Baodi " six lobes are red " Garlic enzyme-deactivating extractive HPLC standard finger-print, the method step is as follows:
(1) sample, kit instrument: totally 10 batches, Baodi " six lobes are red " garlic, use Agilent 1200 type high performance liquid chromatograph, reagent is chromatographically pure or analyzes pure;
(2) preparation of Garlic enzyme-deactivating extract solution to be measured: take Baodi " six lobes are red " garlic bulb 20g, add 25-30ml distilled water, 90 DEG C of enzyme 1h that go out, crushing grinding, add methanol constant volume to 50ml, the centrifugal 15min of 5000 × g, get supernatant through 0.45 μm of filtering with microporous membrane;
(3) preparation of Garlic enzyme-deactivating extract need testing solution: C18 solid phase extraction column, through 5ml methyl alcohol, the activation of 10ml ultrapure water, balances 15min; Solution to be measured crosses post with 4ml/min speed, then uses the drip washing of 5ml ultrapure water, and low vacuum is drained; Again by after 5ml methanol-eluted fractions, under room temperature, low flow velocity nitrogen is concentrated into 0.5-0.8ml, then with methanol constant volume to 1ml, to be measured through 0.45 μm of filtering with microporous membrane;
(4) efficient liquid phase chromatographic analysis: chromatographic column is: EclipsePlusC18,5 μm, 4.6x250mm; Guard column is: EclipsePlusC18Grd, 5 μm, 4.6x12.5mm; Column temperature 35 DEG C; Mobile phase is methyl alcohol and 0.2% aqueous formic acid; Adopt gradient elution mode 0 → 2min → 10min → 20min, methyl alcohol: 60% → 60% → 80% → 80%; Diode array detector determined wavelength 240nm; Sample size 50 μ L; Analyze need testing solution under these conditions, obtain Garlic enzyme-deactivating extractive HPLC fingerprint.
(5) above-mentioned (1) is repeated to (4), " similarity evaluation 2004 editions " software of Chinese Pharmacopoeia Commission is used to analyze 10 batches of Baodis " six lobes are red " Garlic enzyme-deactivating extractive HPLC fingerprint, determine 15 common characteristic peaks, its retention time is respectively: 2.16min, 2.748min, 2.94min, 3.379min, 3.813min, 4.719min, 5.839min, 6.439min, 7.074min, 8.002min, 9.131min, 10.615min, 13.069min, 15.314min, 18.325min, relative standard deviation (RSD) <1%, these common characteristic peaks constitute Baodi " six lobes are red " Garlic enzyme-deactivating extractive HPLC standard finger-print, see Fig. 2.
Software Similarity Measure result that table 1 provides " similarity evaluation 2004 editions ", the similarity of each sample and reference fingerprint all reaches more than 0.99, illustrates that Baodi " six lobes are red " the Garlic enzyme-deactivating extractive HPLC standard finger-print that the present invention determines is accurately.
Table 1 each batch of Baodi " six lobes are red " Garlic enzyme-deactivating extractive HPLC fingerprint Similarity Measure result
Embodiment 3:
Carry out stratographic analysis to the Baodi obtained under different planting " six lobes are red " garlic and other kind garlics, the method step is as follows:
(1) sample, kit instrument: Baodi produces " six lobes are red " garlic, non-Baodi produces " six lobes are red " garlic, Xinjiang White skin garlic, and use Agilent 1200 type high performance liquid chromatograph, reagent is chromatographically pure or analyzes pure;
(2) preparation of Garlic enzyme-deactivating extract solution to be measured: take Baodi " six lobes are red " garlic bulb 20g, add 25-30ml distilled water, 90 DEG C of enzyme 1h that go out, crushing grinding, add methanol constant volume to 50ml, the centrifugal 15min of 5000 × g, get supernatant through 0.45 μm of filtering with microporous membrane;
(3) preparation of Garlic enzyme-deactivating extract need testing solution: C18 solid phase extraction column, through 5ml methyl alcohol, the activation of 10ml ultrapure water, balances 15min; Solution to be measured crosses post with 4ml/min speed, then uses the drip washing of 5ml ultrapure water, and low vacuum is drained; Again by after 5ml methanol-eluted fractions, under room temperature, low flow velocity nitrogen is concentrated into 0.5-0.8ml, then with methanol constant volume to 1ml, to be measured through 0.45 μm of filtering with microporous membrane;
(4) efficient liquid phase chromatographic analysis: chromatographic column is: EclipsePlusC18,5 μm, 4.6x250mm; Guard column is: EclipsePlusC18Grd, 5 μm, 4.6x12.5mm; Column temperature 35 DEG C; Mobile phase is methyl alcohol and 0.2% aqueous formic acid; Adopt gradient elution mode 0 → 2min → 10min → 20min, methyl alcohol: 60% → 60% → 80% → 80%; Diode array detector determined wavelength 240nm; Sample size 50 μ L; Analyze need testing solution under these conditions, obtain Garlic enzyme-deactivating extractive HPLC fingerprint.
Software Similarity Measure result that table 2 provides " similarity evaluation 2004 editions ", wherein S1 is Baodi " six lobes are red " garlic standard finger-print, S2, S3 are that Baodi produces " six lobes are red " garlic, S4, S5 are Xinjiang White skin garlic, and S6, S7 are that non-Baodi produces " six lobes are red " garlic.As can be seen from result of calculation, Baodi " six lobes are red " garlic and standard finger-print similarity the highest, be 0.999, steady quality be described.And Xinjiang White skin garlic and standard finger-print similarity minimum, be 0.956 and 0.957, non-Baodi product " six lobes are red " garlic and standard finger-print similarity are also all lower than Baodi place of production kind, explanation utilizes the method effectively can carry out the true and false and the place of production discriminating of Baodi " six lobes are red " garlic, and can evaluate its interior quality quality comprehensively, exactly;
Claims (2)
1. a construction method for six lobes red Garlic enzyme-deactivating extractive HPLC fingerprint, is characterized in that being undertaken by following step:
(1) preparation of Garlic enzyme-deactivating extract solution to be measured: take the red garlic bulb 20g of six lobes, add 25-30ml distilled water, 90 DEG C of enzyme 1h that go out, crushing grinding, add methanol constant volume to 50ml, the centrifugal 15min of 5000 × g, get supernatant through 0.45 μm of filtering with microporous membrane;
(2) preparation of Garlic enzyme-deactivating extract need testing solution: C18 solid phase extraction column, through 5ml methyl alcohol, the activation of 10ml ultrapure water, balances 15min; Solution to be measured crosses post with 4ml/min speed, then uses the drip washing of 5ml ultrapure water, and low vacuum is drained; Again by after 5ml methanol-eluted fractions, under room temperature, low flow velocity nitrogen is concentrated into 0.5-0.8ml, then with methanol constant volume to 1ml, to be measured through 0.45 μm of filtering with microporous membrane;
(3) efficient liquid phase chromatographic analysis: chromatographic column is: EclipsePlusC18,5 μm, 4.6x250mm; Guard column is: EclipsePlusC18Grd, 5 μm, 4.6x12.5mm; Column temperature 35 DEG C; Mobile phase is methyl alcohol and 0.2% aqueous formic acid; Adopt gradient elution mode 0 → 2min → 10min → 20min, methyl alcohol: 60% → 60% → 80% → 80%; Diode array detector determined wavelength 240nm; Sample size 50 μ L; Analyze need testing solution under these conditions, obtain Garlic enzyme-deactivating extractive HPLC fingerprint;
(4) above-mentioned (1) is repeated to (3), HPLC finger-print is set up to 10 batch of six lobe red Garlic enzyme-deactivating extract, 15 common characteristic peaks are determined by comparative analysis, its retention time is respectively: 2.16min, 2.748min, 2.94min, 3.379min, 3.813min, 4.719min, 5.839min, 6.439min, 7.074min, 8.002min, 9.131min, 10.615min, 13.069min, 15.314min, 18.325min, relative standard deviation (RSD) <1%, these common characteristic peaks constitute six lobes red Garlic enzyme-deactivating extractive HPLC standard finger-print.
2. the red garlic cultivar of six lobes is differentiated and a quality of production control method, it is characterized in that comprising the steps:
(1) preparation of Garlic enzyme-deactivating extract solution to be measured: take the red garlic bulb 20g of six lobes, add 25-30ml distilled water, 90 DEG C of enzyme 1h that go out, crushing grinding, add methanol constant volume to 50ml, the centrifugal 15min of 5000 × g, get supernatant through 0.45 μm of filtering with microporous membrane;
(2) preparation of Garlic enzyme-deactivating extract need testing solution: C18 solid phase extraction column is through 5ml methyl alcohol, the activation of 10ml ultrapure water; Solution to be measured crosses post with 4ml/min speed, then uses the drip washing of 5ml ultrapure water, and low vacuum is drained; After 5ml methanol-eluted fractions, under room temperature, low flow velocity nitrogen is concentrated into 0.5-0.8ml, then with methanol constant volume to 1ml, to be measured through 0.45 μm of filtering with microporous membrane;
(3) efficient liquid phase chromatographic analysis: chromatographic column is: EclipsePlusC18,5 μm, 4.6x250mm; Guard column is: EclipsePlusC18Grd, 5 μm, 4.6x12.5mm; Column temperature 35 DEG C; Mobile phase is methyl alcohol and 0.2% aqueous formic acid; Adopt gradient elution mode 0 → 2min → 10min → 20min, methyl alcohol: 70% → 70% → 80% → 80%; Diode array detector determined wavelength 240nm; Sample size 50 μ L; Analyze need testing solution under these conditions, obtain Garlic enzyme-deactivating extractive HPLC chromatogram;
(4) by step (3), the garlic HPLC chromatogram obtained and six lobes red Garlic enzyme-deactivating extractive HPLC standard finger-print contrast, by the analysis and identification garlic cultivar of chemical component difference contained by it, the place of production or quality.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106771004A (en) * | 2016-12-30 | 2017-05-31 | 南京财经大学 | A kind of method for evaluating Garlic quality |
| CN111337614A (en) * | 2020-04-21 | 2020-06-26 | 中国农业科学院农业质量标准与检测技术研究所 | Metabonomics analysis method for components of garlic bulbs in different growth stages |
| CN111474254A (en) * | 2020-04-14 | 2020-07-31 | 江南大学 | Method for simultaneously detecting alliin and sulfide thereof in black garlic |
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| CN101762663A (en) * | 2009-12-02 | 2010-06-30 | 天津市农业科学院中心实验室 | Preprocessing method for agricultural chemical multiresidue measurement in garlic samples |
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| CN101762663A (en) * | 2009-12-02 | 2010-06-30 | 天津市农业科学院中心实验室 | Preprocessing method for agricultural chemical multiresidue measurement in garlic samples |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106771004A (en) * | 2016-12-30 | 2017-05-31 | 南京财经大学 | A kind of method for evaluating Garlic quality |
| CN106771004B (en) * | 2016-12-30 | 2018-09-11 | 南京财经大学 | A method of evaluation Garlic quality |
| CN111474254A (en) * | 2020-04-14 | 2020-07-31 | 江南大学 | Method for simultaneously detecting alliin and sulfide thereof in black garlic |
| CN111337614A (en) * | 2020-04-21 | 2020-06-26 | 中国农业科学院农业质量标准与检测技术研究所 | Metabonomics analysis method for components of garlic bulbs in different growth stages |
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