CN108205038A - The method for building up of the correlated characteristic finger-print of Dendrobium Chrysotoxum Lindl chemical small molecule ingredient - Google Patents

The method for building up of the correlated characteristic finger-print of Dendrobium Chrysotoxum Lindl chemical small molecule ingredient Download PDF

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CN108205038A
CN108205038A CN201611190855.0A CN201611190855A CN108205038A CN 108205038 A CN108205038 A CN 108205038A CN 201611190855 A CN201611190855 A CN 201611190855A CN 108205038 A CN108205038 A CN 108205038A
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finger
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sample
ingredient
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CN108205038B (en
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邵自星
赵田
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Bluemark Yicheng Hainan Biotechnology Co ltd
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Beijing Lanbiao Yicheng Technology Co Ltd
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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
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Abstract

The invention discloses a kind of method for building up of the correlated characteristic finger-print of the chemical small molecule ingredient of Dendrobium Chrysotoxum Lindl, including：1) chromatography detection is carried out respectively to the sample that sample size is n, obtained using chemical small molecule ingredient schaftoside and/or naringenin as the detection data of reference ingredient；2) finger-print detection is carried out respectively to sample, obtained with the finger-print peak area value of the full chemistry ingredient of Dendrobium Chrysotoxum Lindl；3) with the component content value of schaftoside in chromatographic data and/or naringenin variable in response, the peak area value of ingredients other in finger-print is established into analysis model as independent variable, establishes small molecule ingredient and finger-print linear regression model (LRM)；4) the correlated characteristic finger-print of chemical small molecule ingredient is established to the percentage contribution of regression modeling by variant correlation coefficient index characterization independent variable.The present invention realizes the correlation analysis of chemical small molecule component content, can effectively differentiate, controls and evaluate the quality of Dendrobium Chrysotoxum Lindl medicinal material.

Description

The method for building up of the correlated characteristic finger-print of Dendrobium Chrysotoxum Lindl chemical small molecule ingredient
Technical field
The invention belongs to Chinese medicinal ingredients to test and analyze field, and in particular to a kind of analysis of the finger-print of Dendrobium Chrysotoxum Lindl is built Mould method.
Background technology
The stem of noble dendrobium is a kind of common tonic Chinese herbal medicine, predominantly Dendrobium Sw.Dendrobium Sw is maximum in orchid One category, including multiple kinds such as dendrobium candidum, HERBA DENDROBII, dendrobium, Dendrobium Chrysotoxum Lindl.In the world, there are about More than the 1100 kinds of stem of noble dendrobium, wherein having 76 kinds what China was found.The medicinal history of the stem of noble dendrobium is long.Early in ancient times, the stem of noble dendrobium exists《Legendary god of farming's sheet Grass warp》In be just listed in nourishing top grade, for a long time, with the development of the times, the stem of noble dendrobium is considered as precious medium-height grass by people always Medicine has highly important nourishing effects.Clinically, the stem of noble dendrobium be used to treat a variety of diseases, have strengthen immunity, antioxygen The pharmacological effects such as change, hypoglycemic and inhibition cancer.Due to artificial long-term uncontrolled excavation and the irrational utilization stem of noble dendrobium, wild money Source is reduced increasingly, occurs the phenomenon that some are mixed the spurious with the genuine, adulterate in the market.Further, since stem of noble dendrobium kind is more, product The hybridization of inter-species so that the kind of its nearly edge there are character crossover phenomenon distinguish relatively difficult by classification.Therefore, it is necessary to establish the stem of noble dendrobium Characteristic fingerprint pattern the quality of medicinal material of dendrobe fresh product, dry product is evaluated.Dendrobium Chrysotoxum Lindl (Dendrobium chrysotoxum Lindley)：Stem is upright, meat, spindle, long 6cm~30cm, mid diameter 1.5cm~5cm, 2 section of tool ~5 internodes have the rib of most round blunts, and golden yellow after doing, nearly top has 2 pieces~5 pieces leaves；The not downward sheath to embrace stem of leaf.Always Locusta is sent out from nearly stem top.Tiltedly go out or slightly sagging, up to 20cm；Rachis is sturdy, sparsely grow majority flower；Common peduncle base portion has 4 pieces~5 pieces sheaths；Petal piece is small, film quality, ovum shape lanceolar, long 0.2cm~0.3cm, the anxious point of apex；Bennet and ovary yellow are long Up to 5cm；Flower quality is thick, golden yellow, slightly band fragrance；Middle sepal oblong, long 1.2cm~2cm, middle part width 0.5cm~0.9cm, Apex is slightly blunt, has 7 arteries and veins；Side sepal closely waits big with middle sepal；Calyx capsule is subsphaeroidal, wide about 0.4cm；Petal obovate, waits and is longer than Middle sepal, 2 times of wide about sepal, apex subcircular has about 10 arteries and veins；Lip color is deeper than sepal and petal, nearly kidney shape circle Shape is about 2cm, wide 2.3cm, and apex shallow 2 is split, and how much base portion both sides have a red streak, marginal wavy, above close tomentellate； Lip has the maroon patch of U-shaped sometimes usually in reverse V-shaped protuberance；Stamen column length about 0.5cm；Anther cap is faint yellow, steeple shape.
Chromatographic fingerprinting is a kind of synthesis, quantifiable discriminating means, as a kind of panning mode of global analysis, Reflection is the overall condition of sample, and explains do not have specific aim to the content of single chemical small molecule ingredient.So far, Patent in relation to medicinal dendrobium HPLC finger-prints has 1 (CN200810218494.5), Cheng Jinle etc. to the phenols in the stem of noble dendrobium HPLC fingerprint map analyzings have been carried out with flavones ingredient.The method is suitable for the quality control of this two constituents of phenols and flavonoids System.On the other hand, in chromatographic fingerprinting analytic process, many data are all higher-dimensions, i.e., data include many attributes or Feature for example relating to Dendrobium Chrysotoxum Lindl chromatographic fingerprinting, just can preferably be described Dendrobium Chrysotoxum Lindl, but in practical applications The problem of will facing " dimension disaster " is directly operated to high dimensional data, " dimension disaster " can cause required for modeling process Sample number with dimension increase and exponentially increase.In face of high dimensional data, conventional least square method is no longer applicable in, and is The interpretation of model and the accuracy of prediction are improved, variables choice becomes critically important.How efficiently from numerous variables In filter out several variables to play an important role to independent variable, be that urgent need to resolve is asked when analyzing finger-print Topic.
In addition, all it is the macromoleculars effective components such as detection polysaccharide therein, still, inspection in existing stem of noble dendrobium examination criteria Technology, the instrument requirements height that the macromolecular components such as polysaccharide need are surveyed, domestic existing instrument and equipment usually can not be detected accurately greatly Molecular chaperones, can not the accurate evaluation stem of noble dendrobium quality.
Invention content
The present invention establishes the correlated characteristic finger-print of Dendrobium Chrysotoxum Lindl chemical small molecule ingredient, passes through collection of illustrative plates realization pair The correlation analysis of Dendrobium Chrysotoxum Lindl medicinal material related chemistry small molecule component content.Correlated characteristic using chemical small molecule ingredient refers to Line collection of illustrative plates obtains the information for reflecting medicinal Dendrobium Chrysotoxum Lindl related chemistry small molecule component content.
The purpose of the present invention is what is be achieved through the following technical solutions：
A kind of method for building up of the correlated characteristic finger-print of the chemical small molecule ingredient of Dendrobium Chrysotoxum Lindl, the correlated characteristic The method for building up of finger-print includes：
1) to sample size be n sample carry out chromatography detection respectively, obtain with chemical small molecule ingredient schaftoside and/ Or naringenin, as the detection data with reference to ingredient, the n values are the integer not less than 10；
2) finger-print detection is carried out respectively to sample, obtains the fingerprint image spectral peak face with the full chemistry ingredient of Dendrobium Chrysotoxum Lindl Product value；
3) with the component content value of schaftoside in chromatographic data and/or naringenin variable in response, by finger-print In the peak area values of other ingredients establish analysis model as independent variable, carry out Principle component extraction and return to calculate, establish small point Subconstiuent and finger-print linear regression model (LRM), and obtain regression equation；
4) independent variable is characterized to regression modeling by variant correlation coefficient index (Correlation Coefficient) Percentage contribution, the significance level of the bigger independent variable of corresponding related coefficient is bigger, obtains closely related with small molecule ingredient Fingerprints peak group establishes the correlated characteristic finger-print of chemical small molecule ingredient.
In an embodiment according to the present invention, the chromatography detection described in step 1) is further included at Dendrobium Chrysotoxum Lindl sample Reason method：
The sample treatment includes：
Precision weighs Dendrobium Chrysotoxum Lindl powder, puts in 100ml volumetric flasks, and the first that volume ratio is 75: 25 is added per 1g sample precisions Alcohol-water 50ml is handled 30 minutes, cooled and filtered with 250W power, 40kHz frequency ultrasounds, by filtrate concentrated by rotary evaporation to doing, often 1g Dendrobium Chrysotoxum Lindls powder is accordingly dissolved with the methanol-water that 5ml volume ratios are 75: 25, and supernatant is crossed 0.45 μm of miillpore filter, taken Subsequent filtrate to get Dendrobium Chrysotoxum Lindl small molecule component content determination sample.
In an embodiment according to the present invention, the testing conditions of the chromatography detection described in step 1) include：
Assay chromatographic condition：Grace Allitima C18 chromatographic columns；Mobile phase uses binary gradient elutes system, A phases：0.2% Acetic Acid-Water, B phases：Acetonitrile；Naringenin is measured with wavelength 290nm, schaftoside, reference are measured with wavelength 334nm Wavelength 500nm, 30 DEG C of column temperature；Flow velocity 1.0mL/min, 20 μ L of sample size；Gradient is：
Finger-print chromatographic condition：Grace Allitima C18 chromatographic columns, preferably 250mm × 4.6mm, 5 μm of specifications Chromatographic column；Mobile phase：A phases：0.4% acetic acid+20mmol/L ammonium acetate aqueous solutions, B phases：Acetonitrile；Gradient elution：0~ 12min：2%~15%B phases, 12~35min：15%~24%B phases, 35~45min：24%~36%B phases, 45~60min： 36%~75%B phases, 60~80min：75%~95%B phases；Flow velocity 1.0mL/min；30 DEG C of column temperature；20 μ L of sample size；Detection Wavelength 280nm.
In an embodiment according to the present invention, the analysis model of establishing described in step 2) includes the following steps：
A) when superelevation dimension situation is presented in chromatographic data, using following SIS (SureIndependence Screening) Superelevation dimension is dropped to d (d≤n) dimensions by method by formula l；Wherein, d=n or
Mγ={ 1≤i≤p：|ωi| before being | γ n | a bigger
Formulas I
Wherein, M*={ 1≤i≤p：βi≠ 0 } the subscript collection of nonzero coefficient in true mode is represented；S=| M*| represent non-zero system Several numbers；ω=(ω1, ω2..., ωp)T=XTy；There are as p ＞ n and to some ξ log p=O (nξ), M*With MγBetween have Below in connection with：For any given γ ∈ (0,1), the p member of ω Element is arranged and defined from big to small by absolute value；At this time | γ n | ＜ n choose MγThe corresponding independent variable of middle subscript, reaches dimensionality reduction Purpose；
B) it after SIS method dimensionality reductions, is sieved using SCAD (Smooth Clipped Absolute Deviation) method Select variable.
In an embodiment according to the present invention, the SCAD methods are to calculate to realize by Formula II：
Wherein, penalty pλThe derived function p ' of (β)λ(β) meets ；A ＞ 2, β ＞ 0；Preferably, d=3.7.
Y=(y1, y2..., yn)T∈RnFor response variable vector.With stem of noble dendrobium data instance, there are two respond for each stem of noble dendrobium Variables Sequence (schaftoside (μ g/g), naringenin (μ g/g)).Response variable is influenced by independent variable, and y is connects under normal circumstances Continuous variable.
X=(x1, x2..., xn)TFor the design matrix of n × p, all candidate changes certainly influential on response variable are included Amount.
P is the dimension of sample, and n is sample size.In stem of noble dendrobium data, dimension p is much larger than sample size n sample range, therefore minimum Two, which multiply estimation, is no longer applicable in, and needs the method using variables choice to carry out model estimation.
β=(β1, β2..., βp)TIt is the parameter of a p dimension.
For penalty, λ is adjusting parameter.In Variable Selection, the excellent degree of models fitting It is embodied from for being selected in the balance between the dynamics of variable number punishment by different criterion, and here by directly choosing It takes adjustment parameter to realize, different λ values corresponds to different punishment dynamics.λ is bigger, and the degree of compression is stronger, finally estimates Obtained non-zero parameter is fewer, and it is K folding cross-validation methods to select the most common methods of λ：
K-fold CV：
Generally, K can be taken as 5 or 10.
GCV criterion are a kind of approximate situations when K in CV criterion takes n, are defined as：Wherein, SSEkIt is the residual sum of squares (RSS) of the CV submodels containing k variable, df=trace { P (λ) }.Choosing for final optimal model It selects, the submodel of CV values or GCV values minimum can be taken.
Penalty pλThe derived function p ' of (β)λ(β) meets
Wherein parameter a ＞ 2, β ＞ 0 can pass through Two-Dimensional Generalized cross validation (Generalized Cross- Validation method) is estimated, but calculates more complicated.By the viewpoint combination sunykatuib analysis of Bayes, Fan is proposed A takes 3.7.
SCAD methods meet unbiasedness, openness and continuity, and after suitable regularization parameter is had chosen, SCAD side The estimator that method obtains meets Oracle properties.
The present invention also provides application of the above method in Dendrobium Chrysotoxum Lindl quality control.
The Dendrobium Chrysotoxum Lindl described in above-mentioned Dendrobium Chrysotoxum Lindl quality control can be fresh goods or dry product.
Compared with prior art, the present invention has at least the following advantages：
(a) present invention is carried out variables choice to Dendrobium Chrysotoxum Lindl finger-print, is effectively solved " dimension using SCAD methods The problem of number disaster "；
(b) present invention carries out dimensionality reduction to original finger-print, establishes the correlated characteristic of Dendrobium Chrysotoxum Lindl chemical small molecule ingredient Finger-print explains that specific aim and applicability are stronger to the content of single chemical small molecule ingredient；
(c) present invention realizes chemical small molecule by the correlated characteristic finger-print of Dendrobium Chrysotoxum Lindl chemical small molecule ingredient The correlation analysis of component content can effectively differentiate, control and evaluate the quality of Dendrobium Chrysotoxum Lindl medicinal material.
Description of the drawings
Fig. 1 is Dendrobium Chrysotoxum Lindl finger-print chromatogram；
Fig. 2 a are the test map of schaftoside (No. 1 peak) in Dendrobium Chrysotoxum Lindl；
Fig. 2 b are the test map of naringenin (No. 2 peaks) in Dendrobium Chrysotoxum Lindl.
Specific embodiment
The invention will be further described with reference to the accompanying drawings and examples, and following embodiment is descriptive, is not Limited, it is impossible to protection scope of the present invention is limited with this.
The extracting method of 1 Dendrobium Chrysotoxum Lindl of embodiment
Dendrobium Chrysotoxum Lindl drying sample is taken, is crushed with pulverizer, crosses pharmacopeia sieve (aperture 0.335mm), precision weighs dendrobe powder Last 1.000g (weighing error is no more than 0.2%), is placed in 100ml conical flasks, is separately added into 75% methanol (V of 50mLWater∶ VMethanol=25: 75), take out, filter after ultrasound 30min at room temperature, filtrate concentrated by rotary evaporation is to doing, with 75% methanol solvate (VWater∶VMethanol =25: 75) dissolve, be finally transferred to constant volume in 10ml volumetric flasks, shake up, with 0.45 μm of filtering with microporous membrane to get drumstick stone Dry measure used in former times sample solution.
The chromatographic detection method of 2 dendrobium chrysotoxum extract of embodiment
1. the small molecule component content determination sample extraction process method of Dendrobium Chrysotoxum Lindl：
This product powder (crossing No. three sieves) 1.00g is taken, it is accurately weighed, it puts in 100ml volumetric flasks, accurate plus methanol-water (75: 25) 50ml, is ultrasonically treated (power 250W, frequency 40kHz) 30 minutes, lets cool, and filters, and filtrate concentrated by rotary evaporation is to doing, with 5ml first Alcohol-water (75: 25) dissolves, and supernatant crosses 0.45 μm of miillpore filter, take subsequent filtrate to get.
2. prepared by reference substance solution
Accurately weighed schaftoside 4.10mg, naringenin 4.08mg respectively, are respectively placed in 10ml volumetric flasks, add 75% methanol Dissolved dilution shakes up, as storing solution.It is spare in being refrigerated in 4 DEG C of refrigerators.
It is accurate respectively again to draw a certain amount of reference substance stock solution, add 75% methanol dilution same, it is accurate respectively to prepare the summer Buddhist support glycosides, naringenin reference substance solution.By different dilution ratios, 7 concentration points of prepared composition are diluted.Inject efficient liquid phase Chromatograph.
3. Dendrobium Chrysotoxum Lindl small molecule component content measures chromatographic condition：
Chromatographic condition
In an embodiment according to the present invention, the testing conditions of the chromatography detection described in step 1) include：
Assay chromatographic condition：Grace Allitima C18 chromatographic columns；Mobile phase uses binary gradient elutes system, A phases：0.2% Acetic Acid-Water, B phases：Acetonitrile；Naringenin is measured with wavelength 290nm, schaftoside, reference are measured with wavelength 334nm Wavelength 500nm, 30 DEG C of column temperature；Flow velocity 1.0mL/min, 20 μ L of sample size；Gradient is as shown in table 1.
Finger-print chromatographic condition：Grace Allitima C18 chromatographic columns, preferably 250mm × 4.6mm, 5 μm of specifications Chromatographic column；Mobile phase：A phases：0.4% acetic acid+20mmol/L ammonium acetate aqueous solutions, B phases：Acetonitrile；Gradient elution：0~ 12min：2%~15%B phases, 12~35min：15%~24%B phases, 35~45min：24%~36%B phases, 45~60min： 36%~75%B phases, 60~80min：75%~95%B phases；Flow velocity 1.0mL/min；30 DEG C of column temperature；20 μ L of sample size；Detection Wavelength 280nm.
1 gradient table of table
Fig. 1 show the full ingredients fingerprint chromatogram of Dendrobium Chrysotoxum Lindl, Detection wavelength 280nm；
Fig. 2 a are the test map of schaftoside (No. 1 peak) in Dendrobium Chrysotoxum Lindl；
Fig. 2 b are the test map of naringenin (No. 2 peaks) in Dendrobium Chrysotoxum Lindl.
In 3 Dendrobium Chrysotoxum Lindl of embodiment with the foundation of the correlated characteristic finger-print of schaftoside
1. prepared by stem of noble dendrobium sample solution
Stem of noble dendrobium drying sample is taken, is crushed with pulverizer, crosses pharmacopeia sieve (aperture 0.335mm), precision weighs Dendrobium 1.000g (weighing error is no more than 0.2%), is placed in 100ml conical flasks, is separately added into 50mL75% methanol (VWater∶VMethanol= 25: 75), take out, filter after ultrasound 30min at room temperature, filtrate concentrated by rotary evaporation is to doing, with 75% methanol solvate (VWater∶VMethanol=25: 75) dissolve, be finally transferred to constant volume in 10ml volumetric flasks, shake up, with 0.45 μm of filtering with microporous membrane to get.Table 2 obtains The linear relationship of reference substance schaftoside.
2 reference substance schaftoside linear relationship table of table
2. the method for building up of the correlated characteristic finger-print of Dendrobium Chrysotoxum Lindl schaftoside
Using linear model, due to initial argument p=488, sample size n=13, p ＞ ＞ n, therefore Variable Selection is More important work.This research uses SCAD method selection variables.
SCAD methods screening gained independent variable has 5, R-square 0.7495.
SCAD methods screening gained independent variable and its corresponding coefficient are as shown in table 3, wherein first is classified as selected independent variable volume Number, second is classified as corresponding coefficient, and third is classified as parameter variance, and the 4th is classified as inspection p value.
Table 3：Selected independent variable and its corresponding coefficient obtained by SCAD methods
Using schaftoside as the selection result of response variable：The R of SCAD methods can reach more than 0.74, be selected in independent variable P value is respectively less than 0.05.The meaning of above-mentioned independent variable：The finger-print of Dendrobium Chrysotoxum Lindl n batches sample (n is not less than 10) is according to reservation Finger-print peak area value after time unifying, if X9 is n batches sample (n be not less than 10) in sample size in identical guarantor Stay the 9th finger-print peak area value being aligned under the time.
In general, 5 independents variable can be used as shown in table 4 (being arranged from big to small by related coefficient degree).
Independent variable after 45 screenings of table
X334 X9 X338 X327 X82
The foundation of the correlated characteristic finger-print of 4 Dendrobium Chrysotoxum Lindl naringenin of embodiment
1. prepared by reference substance solution
Accurately weighed naringenin 4.08mg, is placed in 10ml volumetric flasks, adds 75% methanol dissolved dilution, shakes up, as deposit Liquid.It is spare in being refrigerated in 4 DEG C of refrigerators.It is accurate again to draw a certain amount of reference substance stock solution, add 75% methanol dilution, accurately match Naringenin reference substance solution processed.By different dilution ratios, 7 concentration points of prepared composition are diluted, inject high performance liquid chromatography Instrument.Table 5 is the obtained linear relationship of reference substance.
5 reference substance naringenin linear relationship table of table
2. the foundation of Dendrobium Chrysotoxum Lindl naringenin correlated characteristic finger-print
Using linear model, due to initial argument p=488, sample size n=13, p ＞ ＞ n, therefore Variable Selection is More important work.This research uses SCAD method selection variables.
SCAD methods screening gained independent variable has 3, R-square 0.8063.
SCAD methods screening gained independent variable and its corresponding coefficient are as shown in table 6, wherein first is classified as selected independent variable volume Number, second is classified as corresponding coefficient, and third is classified as parameter variance, and the 4th is classified as inspection P values.
Table 6：Selected independent variable and its corresponding coefficient obtained by SCAD methods
Using naringenin as response variable result：The R of SCAD methods can reach more than 0.80, and it is small to be selected in independent variable p value In 0.05.
In general, 3 independents variable can be used as shown in table 7 (being arranged from big to small by related coefficient degree).
Table 7：Independent variable after 3 screenings
X210 X105 X277
The meaning of above-mentioned independent variable：The finger-print of the n batches sample (n is not less than 10) of dendrobium devonianum is according to retention time Finger-print peak area value after alignment, if X210 is the finger of 210th alignment of the n batches sample under identical retention time Line collection of illustrative plates peak area value.
The correlated characteristic finger-print of 5 Dendrobium Chrysotoxum Lindl chemical small molecule ingredient of embodiment is in Dendrobium Chrysotoxum Lindl quality evaluation Using
1. the preparation of test sample determination sample
Precision weighs Dendrobium Chrysotoxum Lindl powder, puts in 100ml volumetric flasks, and the first that volume ratio is 75: 25 is added per 1g sample precisions Alcohol-water 50ml is handled 30 minutes, cooled and filtered with 250W power, 40kHz frequency ultrasounds, by filtrate concentrated by rotary evaporation to doing, often 1g Dendrobium Chrysotoxum Lindls powder is accordingly dissolved with the methanol-water that 5ml volume ratios are 75: 25, and supernatant is crossed 0.45 μm of miillpore filter, taken Subsequent filtrate to get Dendrobium Chrysotoxum Lindl small molecule component content determination sample；
2. chromatography detects
Chromatographic condition：
Chromatographic column：Grace Allitima C18 chromatographic columns (250mm × 4.6mm, 5 μm)；
Mobile phase：A phases：0.4% acetic acid+20mmol/L ammonium acetate aqueous solutions, B phases：Acetonitrile；Gradient elution：0~12min： 2%~15%B phases, 12~35min：15%~24%B phases, 35~45min：24%~36%B phases, 45~60min：36%~ 75%B phases, 60~80min：75%~95%B phases；
Flow velocity, 1.0mL/min；
30 DEG C of column temperature；
20 μ L of sample size；
Detection wavelength 280nm.
Sample preparation methods：
This product powder (crossing No. three sieves) 1.00g is taken, it is accurately weighed, it puts in 100ml volumetric flasks, accurate plus methanol-water (75: 25) 50ml, is ultrasonically treated (power 250W, frequency 40kHz) 30 minutes, lets cool, and filters, and filtrate concentrated by rotary evaporation is to doing, with 5ml first Alcohol-water (75: 25) dissolves, and supernatant crosses 0.45 μm of miillpore filter, take subsequent filtrate to get.
Full ingredients fingerprint is measured with wavelength 280nm when detecting, is pair by obtained full ingredients fingerprint and Fig. 1 According to fingerprint chromatogram carry out similarity comparison；It is up-to-standard that similarity, which is more than 0.85,.
More than, it is merely preferred embodiments of the present invention, but the protection domain invented is not limited thereto, it is any ripe Know those skilled in the art in the technical scope disclosed by the present invention, the change or replacement that can be readily occurred in should all be contained Lid is within protection scope of the present invention.Therefore, protection scope of the present invention should be subject to the protection domain of claims.

Claims (10)

  1. A kind of 1. method for building up of the correlated characteristic finger-print of the chemical small molecule ingredient of Dendrobium Chrysotoxum Lindl, which is characterized in that institute The method for building up for stating correlated characteristic finger-print includes：
    1) chromatography detection is carried out respectively to the sample that sample size is n, obtained with chemical small molecule ingredient schaftoside and/or shaddock Pi Su is as the detection data with reference to ingredient；The n is the integer not less than 10；
    2) finger-print detection is carried out respectively to sample, obtained with the finger-print peak area of the full chemistry ingredient of Dendrobium Chrysotoxum Lindl Value；
    3) with the component content value of schaftoside in chromatographic data and/or naringenin variable in response, by its in finger-print The peak area value of its ingredient establishes analysis model as independent variable, carries out Principle component extraction and return to calculate, establish small molecule into Point with finger-print linear regression model (LRM), and obtain regression equation；
    4) contribution of the independent variable to regression modeling is characterized by variant correlation coefficient index (Correlation Coefficient) Degree, the significance level of the bigger independent variable of corresponding related coefficient is bigger, obtains the fingerprint closely related with small molecule ingredient TuPu method peak group establishes the correlated characteristic finger-print of chemical small molecule ingredient.
  2. 2. the method for building up of the correlated characteristic finger-print of chemical small molecule ingredient as described in claim 1, which is characterized in that Chromatography detection described in step 1) further includes Dendrobium Chrysotoxum Lindl sample treatment：
    The sample treatment includes：
    Precision weighs Dendrobium Chrysotoxum Lindl powder, puts in 100ml volumetric flasks, and it is 75 that volume ratio is added per 1g sample precisions：25 methanol-water 50ml is handled 30 minutes, cooled and filtered with 250W power, 40kHz frequency ultrasounds, by filtrate concentrated by rotary evaporation to dry, the drum per 1g Mallet Dendrobium is accordingly 75 with 5ml volume ratios：25 methanol-water dissolving, supernatant cross 0.45 μm of miillpore filter, take continuous filter Liquid to get Dendrobium Chrysotoxum Lindl small molecule component content determination sample.
  3. 3. the method for building up of the correlated characteristic finger-print of chemical small molecule ingredient as claimed in claim 1 or 2, feature exist In the testing conditions of the chromatography detection described in step 1) include：
    Assay chromatographic condition：Grace Allitima C18 chromatographic columns；Mobile phase uses binary gradient elutes system, A phases： 0.2% Acetic Acid-Water, B phases：Acetonitrile；Naringenin is measured with wavelength 290nm, schaftoside, reference wavelength are measured with wavelength 334nm 500nm, 30 DEG C of column temperature；Flow velocity 1.0mL/min, 20 μ L of sample size；Gradient is：
    Finger-print chromatographic condition：Grace Allitima C18 chromatographic columns, preferably 250mm × 4.6mm, the color of 5 μm of specifications Compose column；Mobile phase：A phases：0.4% acetic acid+20mmol/L ammonium acetate aqueous solutions, B phases：Acetonitrile；Gradient elution：0~12min：2% ~15%B phases, 12~35min：15%~24%B phases, 35~45min：24%~36%B phases, 45~60min：36%~ 75%B phases, 60~80min：75%~95%B phases；Flow velocity 1.0mL/min；30 DEG C of column temperature；20 μ L of sample size；Detection wavelength 280nm。
  4. 4. the method for building up of the correlated characteristic finger-print of chemical small molecule ingredient according to any one of claims 1 to 3, It is characterized in that, the analysis model of establishing described in step 2) includes the following steps：
    A) when superelevation dimension situation is presented in chromatographic data, using following SIS (Sure Independence Screening) method Superelevation dimension is dropped to by d (d≤n) dimensions by Formula II；Wherein, d=n orMγ={ 1≤i≤p：|ωi| before being | γ n | a bigger
    Formula II
    Wherein, M*={ 1≤i≤p：βi≠ 0 } the subscript collection of nonzero coefficient in true mode is represented；S=| M*| represent nonzero coefficient Number；ω=(ω1, ω2..., ωp)T=XTy；There are logp=O (n as p ＞ n and to some ξξ), M*With MγBetween have it is following Contact：For any given γ ∈ (0,1), the p element of ω is pressed Absolute value is arranged and is defined from big to small；At this time | γ n | ＜ n choose MγThe corresponding independent variable of middle subscript；
    B) it after SIS method dimensionality reductions, is screened and become using SCAD (Smooth Clipped Absolute Deviation) method Amount.
  5. 5. according to the method described in claim 4, it is characterized in that, the SCAD methods are to calculate to realize by Formula II：
    Wherein, p is the dimension of sample, and n is sample size；
    β=(β1, β2..., βp)TIt is the parameter of a p dimension；
    As p ＞ n, existWherein λ >=0 is a punishment parameter；For penalty；λ is adjusting parameter, and the adjusting parameter is rolled over cross-validation method by K and selected；
    Penalty pλThe derived function p ' of (β)λ(β) meetsA ＞ 2, β ＞ 0.
  6. 6. method as claimed in claim 5, which is characterized in that a=3.7 in the calculating function of the SCAD methods.
  7. A kind of 7. method of Dendrobium Chrysotoxum Lindl quality evaluation, which is characterized in that by according to any one of claim 1~6 Method obtain correlated characteristic finger-print and test sample finger-print carry out similarity compare, similarity is more than 0.85 It is up-to-standard.
  8. 8. the method for Dendrobium Chrysotoxum Lindl quality evaluation as claimed in claim 7, which is characterized in that the processing method of the test sample For：
    Precision weighs Dendrobium Chrysotoxum Lindl powder, puts in 100ml volumetric flasks, and it is 75 that volume ratio is added per 1g sample precisions：25 methanol-water 50ml is handled 30 minutes, cooled and filtered with 250W power, 40kHz frequency ultrasounds, by filtrate concentrated by rotary evaporation to dry, the drum per 1g Mallet Dendrobium is accordingly 75 with 5ml volume ratios：25 methanol-water dissolving, supernatant cross 0.45 μm of miillpore filter, take continuous filter Liquid to get Dendrobium Chrysotoxum Lindl small molecule component content determination sample.
  9. 9. the method for Dendrobium Chrysotoxum Lindl quality evaluation as claimed in claim 7 or 8, which is characterized in that the chromatography of the test sample Testing conditions are：
    Grace Allitima C18 chromatographic columns, preferably 250mm × 4.6mm, the chromatographic column of 5 μm of specifications；Mobile phase：A phases： 0.4% acetic acid+20mmol/L ammonium acetate aqueous solutions, B phases：Acetonitrile；Gradient elution；Flow velocity 1.0mL/min；30 DEG C of column temperature；Sample introduction Measure 20 μ L；Detection wavelength 280nm；The elution requirement of the gradient elution is：
    0~12min, 2%~15%B phase；
    12~35min, 15%~24%B phase；
    35~45min, 24%~36%B phase；
    45~60min, 36%~75%B phase；
    60~80min, 75%~95%B phase.
  10. 10. the method for the Dendrobium Chrysotoxum Lindl quality evaluation as described in any one of claim 7~9, which is characterized in that the drumstick The stem of noble dendrobium is fresh goods or dry product.
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