CN106124551A - Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance finger printing and method for building up thereof and purposes - Google Patents
Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance finger printing and method for building up thereof and purposes Download PDFInfo
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Abstract
The invention discloses the method for building up of a kind of Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance finger printing, comprise the steps: 1), 95% ethanol will be added carry out supersound extraction after pulverizing for this dry Fructus Citri Sarcodactylis of sample, filtering after extracting solution, gained filtrate is spin-dried for solvent with Rotary Evaporators, obtains crude extract；Take the above-mentioned crude extract of 20mg to be dissolved in the deuterated dimethyl sulfoxide of 0.5mL, as test sample solution；2)、1H NMR tests: utilize nuclear magnetic resonance analyser to use zg30 pulse sequence acquisition above-mentioned test sample solution1H NMR, thus obtain the hydrogen modal data of test sample；After above-mentioned hydrogen modal data is imported software, it is thus achieved that test sample1H NMR finger printing.The present invention provides the Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance finger printing utilizing said method to set up the most simultaneously and carries out the determining method of Fructus Citri Sarcodactylis kind/quality.
Description
Technical field
The present invention relates to a kind of Fructus Citri Sarcodactylis medical material hydrogen nuclear magnetic resonance spectrum (1H-NMR) method for building up and finger printing, belong to medicine
Thing analysis technical field.
Background technology
Fructus Citri Sarcodactylis (Citrus medica L.var.sarcodactylis Swingle), another name citron, citron, pomelo,
Belong to Rutaceae citrus plant, be distributed mainly on Perenniporia martius, China Zhejiang, Jiangxi, Fujian, Guangdong, Guangxi, four
All there is cultivation on the ground such as river, Yunnan.Fructus Citri Sarcodactylis can be divided into multiple kind according to place of origin difference: originates in the medicinal Fructus Citri Sarcodactylis in Guangdong, Guangxi
It is referred to as " FRUCTUS CITRI SARCODACTYLIS "；Originate in the title " gold bergamot " of Jinhua, Zhejiang, originate in the ground such as Sichuan and claim " FRUCTUS CITRI SARCODACTYLIS from Sichuan of China ".Fructus Citri Sarcodactylis has higher medicinal
Be worth, pharmacological evaluation show its there is spasmolytic, the effect such as suppression maincenter has, antiinflammatory and antiallergic；FRUCTUS CITRI SARCODACTYLIS, river Buddhist clinically
Hands and gold bergamot are all used as medicine, but in research process, numerous studies show, different producing area, kind Fructus Citri Sarcodactylis chemical component difference
Substantially, clinical efficacy can be affected.
Along with proton magnetic resonance (PMR) technology (Proton nuclear magnetic resonance,1H-NMR) development,1H-
NMR sensitivity steps up, and nuclear-magnetism technology is the most therewith in pharmaceutical analysis, and this method has been recorded in version " China in 2010
Pharmacopeia " in annex.1H-NMR dactylogram is compared with tradition HPLC dactylogram, it is straightforward to provide structural information, it is not necessary to standard substance,
Need not complicated pre-treatment and pre-separation, there is the characteristic of non-destructive testing.In order to Fructus Citri Sarcodactylis medical material can further obtain abundant profit
With, the quality standard of specification Fructus Citri Sarcodactylis medical material, it is ensured that the quality of Fructus Citri Sarcodactylis medical material, solve the uneven problem of Fructus Citri Sarcodactylis quality of medicinal material.
Summary of the invention
The technical problem to be solved in the present invention is to provide foundation and the fingerprint thereof of a kind of Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance finger printing
Collection of illustrative plates and make usage, the present invention by set up Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance (1H-NMR) finger printing, for differentiating separate sources
Fructus Citri Sarcodactylis medical material and quality thereof provide foundation.
In order to solve above-mentioned technical problem, the present invention provides the method for building up of a kind of Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance finger printing,
Comprise the steps:
1), configuration sample solution:
Using as supplying sample dry Fructus Citri Sarcodactylis (moisture content≤2%) originally to cross 60 mesh sieves after pulverizing, weigh (precision weighs) Fructus Citri Sarcodactylis
Sample powder 8g, (volumetric concentration) the ethanol 300mL that adds 95%；(parameter of ultrasonic technique is: ultrasound wave to carry out supersound extraction 2 times
Power is 400W, and temperature is 30 DEG C)；Each 0.8～1.2h (preferably 1h), filters after united extraction liquid, and gained filtrate is revolved
Turn evaporimeter (controlling evaporating temperature is 50 DEG C) and be spin-dried for solvent (that is, 95% ethanol), obtain crude extract (about 800mg)；
Take the above-mentioned crude extract of 20mg and be dissolved in dimethyl sulfoxide (DMSO-d deuterated for 0.5mL6In), molten as test sample
Liquid；
2)、1H-NMR tests:
Nuclear magnetic resonance analyser (H NMR spectroscopy instrument) is utilized to use zg30 pulse sequence acquisition above-mentioned test sample solution1H-NMR:
Measure temperature 298K (25 DEG C), observing frequency 600.13MHz, spectrum width 5101.1Hz, sampled data points 65536, sweep
Retouching number of times 4 times, TMS (tetramethylsilane) is internal standard；Thus obtain the hydrogen modal data of test sample；
After above-mentioned hydrogen modal data is imported software (for example, MetresNova software), it is thus achieved that test sample1H-NMR refers to
Stricture of vagina collection of illustrative plates.
The improvement of method for building up as the Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance finger printing of the present invention:
By gained1H-NMR finger printing carries out at least one of following 2 kinds of collection of illustrative plates processing modes:
Mode one, sectional integration method: by the test sample of gained1H-NMR finger printing application MetresNova software
Carry out subsection integral, select δ 0.70～8.14 scope1H-NMR figure also carries out subsection integral using every δ 0.04 as 1 unit,
Obtain each chemical shift section corresponding signal peak area value, export data with ASCII fromat；
By each for sample this deduction water peak-to-peak signal and DMSO solvent peak signal after all sections integration and for examination
The number of sample carries out data matrix；According to Similarity Measure principle, utilize Excel 2007 software to carry out similarity analysis, lead
Enter SPSS software and carry out cluster analysis；
Mode two, total peak integration method:
By the test sample of gained1In H-NMR finger printing, nonoverlapping proton signal peak is as total peak, calculates long-pending
Facet is amassed, the total peak area value of gained, imports SPSS software and carries out principal component analysis (PCA).
The further of method for building up as the Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance finger printing of the present invention is improved:
Described step 1) in, 7 supply the places of production of this dry Fructus Citri Sarcodactylis of sample respectively: Zhaoqing Guangdong, Guilin, Jinhua Pinus densiflora
Town, Jinhua Luo Dianzhen, Leshan sichuan, lushan County, sichuan, Jiangjin, Chongqing；
Described step 3):
The sectional integration method of method one, divides 183 integration segment altogether；
The total peak integration method of method two, chooses test sample1Nonoverlapping 20 proton signal peaks in H-NMR collection of illustrative plates
As total peak, calculating integral area, integrating range is as follows:
Obtain 20 total peak area values, import SPSS software and carry out PCA analysis.
The present invention provides the Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance finger printing utilizing said method to set up the most simultaneously and carries out Fructus Citri Sarcodactylis product
The determining method of kind/quality:
With the gold bergamot of gained or FRUCTUS CITRI SARCODACTYLIS from Sichuan of China hydrogen modal data for comparison, sectional integration method is to measuring samples gained hydrogen modal data
Through correlation coefficient >=0.95 that similarity analysis (available Excel 2007 software) obtains and carry out cluster point through SPSS software
It is a class that analysis gathers, and Fructus Citri Sarcodactylis sample the most to be checked is gold bergamot or FRUCTUS CITRI SARCODACTYLIS from Sichuan of China；
With the FRUCTUS CITRI SARCODACTYLIS hydrogen modal data of gained for comparison, sectional integration method to measuring samples gained hydrogen modal data through similarity
Analyze correlation coefficient >=0.95 that (available Excel 2007 software) obtain and carry out cluster analysis to gather through SPSS software be one
Class, Fructus Citri Sarcodactylis sample the most to be checked is FRUCTUS CITRI SARCODACTYLIS.
The improvement of determining method as the Fructus Citri Sarcodactylis quality of the present invention:
PCA analysis result is: 9 total peak δ 2.870～2.910, δ 3.000～3.050, δ 3.050～3.090, δ
4.250～4.290, δ 6.200～6.260, δ 6.570～6.630, δ 8.000～8.030, δ 3.240～3.260 and δ 3.090
～the peak area of 3.140 is relatively large, Fructus Citri Sarcodactylis quality is relatively good.
The beneficial effects are mainly as follows:
The present invention set up Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance (1H-NMR) finger printing, for differentiate separate sources Fructus Citri Sarcodactylis medical material and
Its quality provides foundation.Utilize the Fructus Citri Sarcodactylis of nmr determination different sources, gained free damping signal is imported
MestReNova software carries out subsection integral, carry out respectively after the segmented Integral Processing of NMR data Semblance, cluster analysis and
PCA is evaluated.Result display gold bergamot and the correlation coefficient of FRUCTUS CITRI SARCODACTYLIS from Sichuan of China all more than 0.95, and with the phase of FRUCTUS CITRI SARCODACTYLIS
Seemingly spending relatively low, correlation coefficient is all below 0.86；Limettin relative amount in gold bergamot and FRUCTUS CITRI SARCODACTYLIS from Sichuan of China is more than FRUCTUS CITRI SARCODACTYLIS；
Cluster analysis result also illustrates that FRUCTUS CITRI SARCODACTYLIS can be clearly separated with gold bergamot and FRUCTUS CITRI SARCODACTYLIS from Sichuan of China, and gold bergamot compares phase with FRUCTUS CITRI SARCODACTYLIS from Sichuan of China
Seemingly；Principal component analysis obtains four main constituents, and its contribution rate of accumulative total reaches 95.430%, first principal component is main and δ 2.870～
2.910, δ 3.000～3.050, δ 3.050～3.090, δ 4.250～4.290, δ 6.200～6.260, δ 6.570～6.630 etc.
Six integration segment peak heights positive correlations, the 2nd main constituent mainly reflects the information of Limettin H-4 peak (δ 8.000～8.030), the
3 and the 4th main constituent factor reflects the information at δ 3.240～3.260 and δ 3.090～3.140 peak respectively.The method can quickly letter
Just distinguish the Fructus Citri Sarcodactylis of different cultivars, can be that quality control and the use of Fructus Citri Sarcodactylis provides reference.
Accompanying drawing explanation
Below in conjunction with the accompanying drawings the detailed description of the invention of the present invention is described in further detail.
Fig. 1(a)It is the typical case of 7 batches of separate sources Fructus Citri Sarcodactylis sample solutions1H-NMR finger printing；
(b)δ 7.5～8.5 partial enlarged drawing；
A. Zhaoqing Guangdong；B. Guilin；C. Jinhua Pinus densiflora town；D. Jinhua Luo Dianzhen；E. Leshan sichuan；F. Sichuan reed
Mountain；G. Jiangjin, Chongqing；H. Limettin.
Fig. 2 is the molecular structural formula of Limettin.
Fig. 3 is cluster analysis dendrogram.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This.By inventive feature, technological means and the specific purposes reached, function can be further appreciated that, resolve the present invention's
Advantage and spirit, be further understood the detailed description of the present invention with detailed description of the invention by below in conjunction with accompanying drawing.
Embodiment 1,
1 experimental section
1.1 key instruments, reagent and material
Key instrument: (Switzerland Bruker is public for Bruker AVANCE III 600 type superconduction Fourier transform H NMR spectroscopy instrument
Department)；AS7240B type ultrasonic device (Tianjin Ao Tesaiensi Instrument Ltd.)；BSl24S type 1,/10,000 electronic balance (Germany
Sartorius)；RZ-52B Rotary Evaporators (Asia, Shanghai is flourish)；
Main agents and material: distilled water, 95% ethanol (analytical pure, Beijing Chemical Plant), DMSO-d6(Cambridge
Isotope company).The Fructus Citri Sarcodactylis (seeing table 1) of different sources.
1.2 the configuration of test sample solution
Crossing 60 mesh sieves after pulverizing for this dry Fructus Citri Sarcodactylis (moisture content≤2%) of sample, precision weighs Fructus Citri Sarcodactylis sample powder 8g, adds
95% ethanol 300mL；Carry out supersound extraction 2 times (parameter of ultrasonic technique is: ultrasonic power is 400W, and temperature is 30 DEG C),
1h, filters to get filtrate after united extraction liquid every time, is spin-dried for solvent with Rotary Evaporators (controlling evaporating temperature is 50 DEG C), obtains and slightly soak
Cream (about 800mg).Take the above-mentioned crude extract of 20mg, be dissolved in dimethyl sulfoxide (DMSO-d deuterated for 0.5mL6In).
1.3 1H-NMR test condition
Utilize the test sample solution of nuclear magnetic resonance analyser (H NMR spectroscopy instrument) employing zg30 pulse sequence acquisition 1.2 preparation1H-
NMR spectra.
Measure temperature 298K (25 DEG C), observing frequency 600.13MHz, spectrum width 5101.1Hz, sampled data points 65536, sweep
Retouching number of times 4 times, tetramethylsilane (TMS) is internal standard.Thus obtain the hydrogen modal data of test sample；
After above-mentioned hydrogen modal data is imported software (for example, MetresNova software), particularly as follows: will detect through NMR instrument
Arrive1H-NMR free damping (FID) signal imports MestReNova software and carries out automatic fourier transform, manually adjusts phase place
With baseline, on the basis of internal standard substance TMS, correct chemical shift, i.e. obtain test sample1H-NMR finger printing.Such as Fig. 1 institute
Show.
1.4 1H-NMR collection of illustrative plates processes
Sectional integration method: application MetresNova software carries out subsection integral, selects δ 0.70～8.14 scope1H-NMR
Figure also carries out subsection integral using every δ 0.04 as 1 unit, obtains each chemical shift section corresponding signal peak area
Value, exports data with ASCII fromat；This test obtains 183 sections of integrations altogether, and (water peak-to-peak signal δ 3.26～3.34 and DMSO is molten for deduction
Agent peak-to-peak signal δ 2.50～2.54).The data matrix (183 variable × 7 samples) that will obtain, according to Similarity Measure principle,
Utilize Excel 2007 software to carry out similarity analysis, import SPSS software and carry out cluster analysis.
Remarks illustrate: the place of production of above-mentioned 7 samples respectively: A. Zhaoqing Guangdong；B. Guilin；C. Jinhua Pinus densiflora town；
D. Jinhua Luo Dianzhen；E. Leshan sichuan；F. lushan County, sichuan；G. Jiangjin, Chongqing；
Total peak integration method: choose nonoverlapping 20 proton signal peaks in Fig. 1 and, as total peak, calculate integral area,
Integrating range is shown in Table 3, it is thus achieved that 20 total peak area values, imports SPSS software and carries out PCA analysis.
1.5 methodological study
1.5.1 precision test
Take with a Fructus Citri Sarcodactylis test sample solution (being prepared according to 1.2), above-mentioned1H-NMR presses 1.3 test conditions
Measuring finger printing the most continuous 6 times, with MetresNova software processes collection of illustrative plates, the integration method under using 1.4 obtains one and is
Row relative peak area integrated value.Calculate the correlation coefficient between 6 groups of data, analytical tool precision.Sectional integration method calculates phase
Like coefficient results all more than 0.99, show that instrument is accurately.
1.5.2 replica test
By configuring same batch Fructus Citri Sarcodactylis test sample solution 6 parts under 1.2 methods, by 1.3 lower condition tests1H-NMR
Finger printing, processes collection of illustrative plates by 1.4 lower methods, calculates the correlation coefficient between 6 groups of data relative peak area integrated values, investigates
Its repeatability situation.Similarity coefficient result of calculation, all more than 0.99, shows that the repeatability of the method is good.
1.5.3 stability test
Take with a Fructus Citri Sarcodactylis test sample solution, respectively 0,1,2,3,4,5d by 1.3 under condition test1H-NMR refers to
Stricture of vagina collection of illustrative plates, with the correlation coefficient calculated under 1.4 between 6 groups of data relative peak area integrated values, similarity coefficient result of calculation is equal
More than 0.99, show that test sample solution is stable in 6d.
2 results and analysis
2.1 1H-NMR finger printing
By 7 batches of Fructus Citri Sarcodactylis samples available after MetresNova software processes collection of illustrative plates1H-NMR finger printing (Fig. 1).Logical
Cross analysis directly perceived, it can be seen that FRUCTUS CITRI SARCODACTYLIS (Zhaoqing Guangdong. Guilin), gold bergamot (Pinus densiflora town, Jinhua. Jinhua Luo Dianzhen),
3 different cultivars Fructus Citri Sarcodactylis extracts such as FRUCTUS CITRI SARCODACTYLIS from Sichuan of China (Leshan sichuan, lushan County, sichuan, Jiangjin, Chongqing)1The profile of H-NMR substantially phase
With, all contain flavonoid, saccharide, organic acid and fatty acid composition.But the intensity of some signal peaks of different bases exists
Different, as in the region of δ 3.3～3.6, FRUCTUS CITRI SARCODACTYLIS signal peak strength in this region is markedly less than other 2
Kind, shows that 3 interracial chemical composition contents of base have certain difference.
There are some researches show that Limettin (also known as citropten .) (seeing Fig. 2) content in Fructus Citri Sarcodactylis medical material is higher, be gold
A main secondary metabolite in Fructus Citri Sarcodactylis, FRUCTUS CITRI SARCODACTYLIS and FRUCTUS CITRI SARCODACTYLIS from Sichuan of China, and modern pharmacology experiment prove Limettin be
One of principle active component of Fructus Citri Sarcodactylis medical material, so can be using citropten. as one of the index of Fructus Citri Sarcodactylis evaluation of medical materials' quality, this
This compound is isolated in invention from gold bergamot extract, and its hydrogen spectrogram sees Fig. 1, and concrete hydrogen modal data is as follows:1H-NMR
(DMSO-d6, 600.13MHz) δ: 8.02 (1H, d, J=9.7Hz, H-4), 6.53 (1H, d, J=2.1Hz, H-6), 6.20 (1H,
D, J=9.7Hz, H-3), 6.62 (1H, d, J=2.1Hz, H-8), 3.87 and 3.91 (each 3H, s, OCH3 × 2), its spectral data
Basically identical with document, can identify that this compound is 5,7 one dimethyl coumarins (Limettin).?1In H-NMR, resonance
Peak area or peak height are directly proportional to the proton number of this formant of generation, and this is quantitative foundation, available characteristic peak relative amount
Method carries out macroscopic analysis to the content of composition.In Limettin hydrogen is composed, H-4 (δ 8.02) peak of Limettin reaches baseline and divides
From and both sides baseline smooth (seeing Fig. 1 b), not by other signal disturbing, meet quantitative principle, therefore, selected δ 8.02 peak is lemon
The quantitative peak of lemon lactone relative amount, can intuitively find out from figure Limettin relative amount in gold bergamot and FRUCTUS CITRI SARCODACTYLIS from Sichuan of China than
FRUCTUS CITRI SARCODACTYLIS is many.
2.2 statistical analysis
2.2.1 similarity evaluation
The Fructus Citri Sarcodactylis medical material of 7 batches of separate sources is prepared as test sample solution according to the method under 1.2, under using 1.3
Condition test above sample hydrogen spectrum, use sectional integration method process1H-NMR finger printing, to the NMR integration obtained
Area data matrix carries out Similarity Measure.
Select the collection of illustrative plates of Pinus densiflora town, Jinhua Fructus Citri Sarcodactylis as reference fingerprint, calculate the correlation coefficient value of 7 batch samples, result
The correlation coefficient of display Jinhua Pinus densiflora town Fructus Citri Sarcodactylis and Jinhua Luo Dianzhen Fructus Citri Sarcodactylis and 3 FRUCTUS CITRI SARCODACTYLIS from Sichuan of China is all between 0.9529～0.9981
(seeing table 1), and Jinhua Luo Dianzhen Fructus Citri Sarcodactylis is relatively low with the similarity of 2 FRUCTUS CITRI SARCODACTYLISs, similarity is 0.7969～0.8581, prompting
Gold bergamot is closely similar with the chemical composition of FRUCTUS CITRI SARCODACTYLIS from Sichuan of China, and the chemical composition of gold bergamot and FRUCTUS CITRI SARCODACTYLIS there is some difference.
The place of production, source of 1,7 batches of Fructus Citri Sarcodactylis of table and similarity evaluation result (Sample source and the
similarity of 7batches raw materials of Citrus medica L.var.sarcodactylis
Swingle)
2.2.2 cluster analysis and principal component analysis evaluation
2.2.2.1 cluster analysis
Use sectional integration method, as initial data after the integrating peak areas value normalization of different sources Fructus Citri Sarcodactylis medical material,
SPSS19.0 software carries out hierarchial-cluster analysis to related data, and average connection between employing group, with included angle cosine for sample phase
Like the range formula of degree, cluster analysis result is shown in Fig. 3.From the figure 3, it may be seen that Fructus Citri Sarcodactylis medical material is divided into 2 big classes, a class includes FRUCTUS CITRI SARCODACTYLIS
A (Zhaoqing Guangdong) and B (Guilin)；An other class class includes gold bergamot C (Pinus densiflora town, Jinhua), D (Jinhua Luo Dianzhen) and river
Fructus Citri Sarcodactylis E (Leshan sichuan), F (lushan County, sichuan), G (Jiangjin, Chongqing).Therefore, between each place of production Fructus Citri Sarcodactylis obtained by cluster analysis
Dependency consistent with the result of similarity analysis, be mutually authenticated between them.
2.1.2.2 1H-NMR has peak principal component analysis
PCA is a kind of widely used multivariate statistical method, is used for simplifying data, quick implementation pattern or
The visualization identification of relation.Initial data is standardized processing, with eigenvalue and the contribution of main constituent by SPSS19.0 software
Rate is foundation, and 20 total peaks of 7 different sources Fructus Citri Sarcodactylis medical materials are carried out principal component analysis, the results are shown in Table 2 and table 3.
Table 2, the main constituent eigenvalue of extraction and contribution rate (The characteristic value and
contribution of principal components)
Table 3, main constituent Factor load-matrix (Loading matrix of principal component factor)
As shown in Table 2, the extraction principle of main constituent number is its characteristic of correspondence value more than 1, thus take front 4 as main one-tenth
Point, its contribution rate of accumulative total reaches 95.43%, and it represents the quantity of information of the 95.43% of studied spectral peak.Wherein, the first main one-tenth
Dtex value indicative is 11.281, and contribution rate is 56.406%, is the most important components group of Fructus Citri Sarcodactylis medical material.As can be seen from Table 3, the 1st
The main constituent factor and 3～5,15,18, No. 19 characteristic peak high-positive correlations, point out δ 2.870～2.910, δ 3.000～3.050, δ
3.050～3.090, δ 4.250～4.290, these six integration segment peaks of δ 6.200～6.260, δ 6.570～6.630 are to the most same
The differentiation evaluation contribution of product is bigger；The 2nd main constituent factor and No. 20 characteristic peak high-positive correlations, illustrate the 2nd main constituent reflection
The mainly information at δ 8.000～8.030 peak, the i.e. the 2nd main constituent mainly reflects the information at Limettin H-4 peak (seeing Fig. 2)；
3rd and the 4th main constituent factor respectively with No. 7 and No. 6 characteristic peak high-positive correlations, point out they reflect respectively δ 3.240～
The information at 3.260 and δ 3.090～3.140 peaks.The characteristic peak of above-mentioned high-positive correlation has phase in the quality control of Fructus Citri Sarcodactylis medical material
To important effect.
In sum, use cluster analysis and principal component analysis, can realize quickly analyzing, display one's respective advantages, test mutually
Demonstrate,proving and supplement, multi-angle comprehensive passes judgment on the quality of Fructus Citri Sarcodactylis medical material.
Embodiment 2, by following 3 kinds of Fructus Citri Sarcodactylis in the previously known place of production: Fructus Citri Sarcodactylis 1. (place of production is Pinus densiflora town, Jinhua), Fructus Citri Sarcodactylis are 2.
(place of production is Leshan sichuan), Fructus Citri Sarcodactylis 3. (place of production is Zhaoqing Guangdong), step 1.2～step 1.4 according to embodiment 1 are examined
Survey,
1. acquired results is respectively as follows: Fructus Citri Sarcodactylis is 0.96 with Fructus Citri Sarcodactylis 2. correlation coefficient, and cluster analysis is classified as a class；3. Fructus Citri Sarcodactylis returns
For an other class.
Similarity evaluation and the cluster analysis result of the above results with embodiment 1 step 2.2 gained are compared, comparison
Acquired results is: 2. 1. Fructus Citri Sarcodactylis be gold bergamot or FRUCTUS CITRI SARCODACTYLIS from Sichuan of China with Fructus Citri Sarcodactylis；3. Fructus Citri Sarcodactylis is FRUCTUS CITRI SARCODACTYLIS.
According to above-mentioned comparison result, we learn: use the present invention method can effectively to gold bergamot or FRUCTUS CITRI SARCODACTYLIS from Sichuan of China with wide
Fructus Citri Sarcodactylis makes a distinction.
Finally, in addition it is also necessary to be only several specific embodiments of the present invention it is noted that listed above.Obviously, this
Bright it is not limited to above example, it is also possible to have many deformation.Those of ordinary skill in the art can be from present disclosure
The all deformation directly derived or associate, are all considered as protection scope of the present invention.
Claims (6)
1. the method for building up of Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance finger printing, is characterized in that comprising the steps:
1), configuration sample solution:
Using as supplying sample dry Fructus Citri Sarcodactylis originally to cross 60 mesh sieves after pulverizing, weigh Fructus Citri Sarcodactylis sample powder 8g, add 95% ethanol 300mL；
Carry out supersound extraction 2 times；Each 0.8～1.2h, to filter after united extraction liquid, gained filtrate is spin-dried for solvent with Rotary Evaporators,
Obtain crude extract；
Take the above-mentioned crude extract of 20mg to be dissolved in the deuterated dimethyl sulfoxide of 0.5mL, as test sample solution；
2)、1H-NMR tests:
Nuclear magnetic resonance analyser is utilized to use zg30 pulse sequence acquisition above-mentioned test sample solution1H-NMR:
Mensuration temperature 298K, observing frequency 600.13MHz, spectrum width 5101.1Hz, sampled data points 65536, scanning times 4 times,
TMS is internal standard；Thus obtain the hydrogen modal data of test sample；
After above-mentioned hydrogen modal data is imported software, it is thus achieved that test sample1H-NMR finger printing.
The method for building up of Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance finger printing the most according to claim 1, is characterized in that:
By gained1H-NMR finger printing carries out at least one of following 2 kinds of collection of illustrative plates processing modes:
Mode one, sectional integration method: by the test sample of gained1H-NMR finger printing application MetresNova software is carried out point
Duan Jifen, selects δ 0.70～8.14 scope1H-NMR figure also carries out subsection integral using every δ 0.04 as 1 unit, obtains each
Chemical shift section corresponding signal peak area value, exports data with ASCII fromat；
By each for sample this deduction water peak-to-peak signal and DMSO solvent peak signal after all sections integration and for sample this
Number carry out data matrix；According to Similarity Measure principle, utilize Excel 2007 software to carry out similarity analysis, import
SPSS software carries out cluster analysis；
Mode two, total peak integration method:
By the test sample of gained1In H-NMR finger printing, nonoverlapping proton signal peak is as total peak, calculates integration face
Long-pending, the total peak area value of gained, import SPSS software and carry out principal component analysis.
The method for building up of Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance finger printing the most according to claim 1 and 2, is characterized in that:
Described step 1) in, 7 places of production for this dry Fructus Citri Sarcodactylis of sample respectively: Zhaoqing Guangdong, Guilin, Pinus densiflora town, Jinhua,
Jinhua Luo Dianzhen, Leshan sichuan, lushan County, sichuan, Jiangjin, Chongqing；
Described step 3):
The sectional integration method of method one, divides 183 integration segment altogether；
The total peak integration method of method two, chooses test sample1Nonoverlapping 20 proton signal peak conducts in H-NMR collection of illustrative plates
Total peak, calculates integral area, and integrating range is as follows:
Obtain 20 total peak area values, import SPSS software and carry out PCA analysis.
4. utilize the Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance finger printing that method as described in claims 1 to 3 is arbitrary is set up.
5. utilize as described in claims 1 to 3 is arbitrary method foundation Fructus Citri Sarcodactylis hydrogen nuclear magnetic resonance finger printing carry out Fructus Citri Sarcodactylis kind/
The determining method of quality, is characterized in that:
With the gold bergamot of gained or FRUCTUS CITRI SARCODACTYLIS from Sichuan of China hydrogen modal data for comparison, sectional integration method to measuring samples gained hydrogen modal data through phase
Analyze, like degree, correlation coefficient >=0.95 that obtains and carry out cluster analysis to gather through SPSS software be a class, Fructus Citri Sarcodactylis sample the most to be checked
For gold bergamot or FRUCTUS CITRI SARCODACTYLIS from Sichuan of China；
With the FRUCTUS CITRI SARCODACTYLIS hydrogen modal data of gained for comparison, sectional integration method to measuring samples gained hydrogen modal data through similarity analysis
Correlation coefficient >=0.95 that obtains and carry out cluster analysis to gather through SPSS software be a class, Fructus Citri Sarcodactylis sample the most to be checked is wide Buddhist
Hands.
The determining method of Fructus Citri Sarcodactylis quality the most according to claim 5, is characterized in that:
PCA analysis result is: 9 total peak δ 2.870～2.910, δ 3.000～3.050, δ 3.050～3.090, δ 4.250～
4.290, δ 6.200～6.260, δ 6.570～6.630, δ 8.000～8.030, δ 3.240～3.260 and δ 3.090～3.140
Peak area relatively large, Fructus Citri Sarcodactylis quality is relatively good.
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| CN107703173A (en) * | 2017-06-23 | 2018-02-16 | 孟扬 | A kind of identification apparatus and method of the rare species timber based on 1H NMR datas storehouse |
| CN115406920A (en) * | 2022-07-26 | 2022-11-29 | 中轻检验认证有限公司 | Method for quantitatively detecting content of limonene in orange oil by utilizing nuclear magnetic resonance hydrogen spectrum |
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| CN102147378A (en) * | 2011-02-28 | 2011-08-10 | 周至譓 | Method for analyzing traditional Chinese medicine dimethyl sulfoxide extracting solution by nuclear magnetic resonance technology |
| CN103399028A (en) * | 2013-07-24 | 2013-11-20 | 厦门大学 | Detection method of juice |
| CN104297277A (en) * | 2013-07-15 | 2015-01-21 | 天士力制药集团股份有限公司 | 1H-NMR fingerprint spectrum of compound red sage root dropping pill |
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| CN102147378A (en) * | 2011-02-28 | 2011-08-10 | 周至譓 | Method for analyzing traditional Chinese medicine dimethyl sulfoxide extracting solution by nuclear magnetic resonance technology |
| CN104297277A (en) * | 2013-07-15 | 2015-01-21 | 天士力制药集团股份有限公司 | 1H-NMR fingerprint spectrum of compound red sage root dropping pill |
| CN103399028A (en) * | 2013-07-24 | 2013-11-20 | 厦门大学 | Detection method of juice |
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| CN107703173A (en) * | 2017-06-23 | 2018-02-16 | 孟扬 | A kind of identification apparatus and method of the rare species timber based on 1H NMR datas storehouse |
| CN115406920A (en) * | 2022-07-26 | 2022-11-29 | 中轻检验认证有限公司 | Method for quantitatively detecting content of limonene in orange oil by utilizing nuclear magnetic resonance hydrogen spectrum |
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