CN109959751A - Water-soluble natural product thin layer quantitative image recognition detection method - Google Patents
Water-soluble natural product thin layer quantitative image recognition detection method Download PDFInfo
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Abstract
The invention discloses a kind of water-soluble natural product thin layer quantitative image recognition detection methods, comprising: (1) sample to be tested containing water-soluble natural product carries out point sample on chromatographic sheet；(2) thin-layer chromatography is carried out to the chromatographic sheet after point sample using the first solvent and the second solvent to be unfolded step by step；First solvent is ethyl acetate/ethanol/water/acetic acid composition expansion system, and second solvent is chloroform/ethyl acetate/formic acid composition expansion system；(3) it is developed the color using coloring agent system to the chromatographic sheet after expansion；(4) Image Acquisition is carried out to the chromatographic sheet after colour developing and the content of water-soluble natural product is obtained according to the relationship between image and water-soluble natural product assay.Method of the invention is easy to operate, does not need special instrument.Method of the invention is quantitative good, and accuracy is high.Method test speed of the invention is fast, can be measured simultaneously with multiple samples.
Description
Technical field
The invention belongs to natural products and food and medicine field, and in particular to a kind of water-soluble natural product thin layer is quantitatively schemed
As recognition detection method.
Background technique
It is active constituent important in plant that water-soluble natural product, which includes flavonoids, flavonoid glycoside, anthocyanin, saponin(e, tool
There are bacteriostasis, preservation, anti-oxidant, anti-aging, antibacterial, antiviral, reducing blood lipid, anticancer and other effects.Flavonoids, flavonoid glycoside, anthocyanin
Water-soluble natural product has excellent antioxidation, can generate stable compound by Scavenger of ROS cluster or its reaction
Mode active balance people's interior free yl, and its metal ion chelant ability can terminate free chain reaction.A large amount of body
Interior and experiment in vitro and epidemiologic data show that saponin(e water-soluble natural product has anti-inflammatory, lipid-loweringing, anti-diabetic and painstaking effort
Biology and the pharmacological actions such as pipe disease (CVD) prevention are led in high-tech such as pharmacy, biochemistry, daily use chemicals, food and fine chemistry industries
Domain has broad application prospects.Common water-soluble natural product detection method has spectrophotometry method, liquid phase method, still
Dedicated instrument is needed, is operated relatively cumbersome.
Nearest computer image processing technology is grown rapidly, and the quantitative analysis of image is feasible.Using ImageJ as representative
Image processing program be widely used in life science field, such as electrophoretic band quantitative analysis, cell count, institutional framework
Quantitative Treatment etc..ImageJ is the public sphere Java image processing program of NIH exploitation.It can have Java any
1.4 or more highest version virtual machine computer on as online applet or can download application program operation.Downloadable distribution
Version is suitable for Windows, Mac OS, Mac OS X and Linux.It can show, edit, and analyze, processing, save and print 8
Position, 16 and 32 bit images.It can read many picture formats, including TIFF, GIF, JPEG, BMP, DICOM, FITS and
"raw".It supports " storehouse ", a series of image of shared single windows.It is multithreading, therefore can be with other operations simultaneously
Row executes the time-consuming operation of such as image file reading etc.It can calculate the area and pixel primary system that user defines selection
Meter.It can measure distance and angle.It can create density histogram and line profile figure.It supports standard picture to handle function
Can, such as contrast operation sharpens, smoothly, edge detection and median filtering.It can carry out geometric transformation, such as scale, rotation and
Overturning.All analyses and processing function can use under any amplification coefficient.The program supports any number of window simultaneously
(image), is only limited by free memory.Spatial calibration can be used for providing the dimensional measurement of real world in millimeters.Also
It can carry out density or gamma calibration.ImageJ is designed using open architecture, provides scalability by java plug-in.It can be with
Customized acquisition, analysis and processing plug-in unit are developed using the built-in editing machine and Java compiler of ImageJ.What user write inserts
Part can solve substantially any image procossing or problem analysis.
The tlc analysis of natural products is a kind of common high efficiency technical, but traditional thin layer quantitative analysis needs are dedicated
Instrument, using inconvenience.Therefore newest quantitative analysis with computer means are based on, the thin layer for carrying out flavonoids polyphenol is quantitative
Image recognition detection can simplify operation, effectively widen use scope.Have at present and some has been lived using image recognition
Property component quantifying detection research.Such as application number 200510115774.X patent document, it discloses a kind of based on image procossing
The thin layer chromatography quantitative analysis method of technology, comprising steps of (1) takes pictures to form digital picture to sample lamellae is loaded with；(2) right
Shoot image preprocessing: lens distortion calibration filters out noise；(3) it is uniformly distributed using point sample, fore-and-aft distance equipartition principle determines
Control point；(4) according to control point interpolation method structural map as background；(5) background is removed from lens distortion calibration image, according to back
Scape normalizes pixel brightness；(6) (7) are split to thin layer image to integrate the area grayscale after segmentation, quantitatively
Analysis.But this method is only described the General Principle of image analysis, not can be used directly determining for specific natural products
Amount detection.The patent document of application number 201510163742.0 discloses a kind of this resistance to sugared content measuring method of Morinda officinalis, utilizes
Trapezoidal integration integrates chromatographic peak, carries out quantitative analysis according to peak area and sample concentration.But this method is only limitted to one
The measurement of kind specific sugar.The patent document of application number 201810451846.5 discloses a kind of method for identifying honey authenticity, uses
ImageJ software analyzes the oligosaccharides in honey sample.But this method is only limitted to the analysis of a kind of sugar.Therefore it develops and is applicable in
In different types of thin layer image recognition quantitative analysis method, there is important meaning for the quick analysis and identification of natural products
Justice.
Summary of the invention
It is easy that the present invention provides a kind of water-soluble natural product thin layer quantitative image recognition detection method and processes, does not need
Special instrument, it is widely applicable.
A kind of water-soluble natural product thin layer quantitative image recognition detection method, comprising:
(1) sample to be tested containing water-soluble natural product is subjected to point sample on chromatographic sheet；
(2) thin-layer chromatography is carried out to the chromatographic sheet after point sample using the first solvent and the second solvent to open up step by step
It opens；First solvent is ethyl acetate/ethanol/water/acetic acid composition expansion system, and second solvent is three chloromethanes
Alkane/ethyl acetate/formic acid composition expansion system；
(3) it is developed the color using coloring agent system to the chromatographic sheet after expansion；
(4) Image Acquisition is carried out to the chromatographic sheet after colour developing, according between image and water-soluble natural product assay
Relationship, obtain the content of water-soluble natural product.
Specifically, a kind of water-soluble natural product thin layer quantitative image recognition detection method is based on computer picture recognition
Simultaneous Quantitative Analysis is carried out to different water-soluble natural products (flavonoids, flavonoid glycoside, anthocyanin, saponin(e) and its mixture,
The following steps are included:
(1) using ethyl acetate/ethanol/water/acetic acid and chloroform/ethyl acetate/formic acid solvent system to difference
Major class natural products carries out thin-layer chromatography to be unfolded step by step；
(2) thin-layer chromatography colour developing is carried out to different water-soluble natural products using unified coloring agent system；
(3) to water-soluble natural product thin-layer chromatography image electronization and quantitative analysis, show that final water-soluble natural produces
Object thin layer quantitative result.
In the present invention, water-soluble natural product before detection, can use solvent dissolution, be formulated as water-soluble natural product
Then sample solution carries out Development of Thin-Layer Chromatography again；The water-soluble natural Product samples solution concentration is 0.01-10mg/
mL；Further preferably 0.5-5mg/mL.When using thin-layer chromatography, point sample amount is 0.1-10 microlitres；Preferably 0.1~1 microlitre.
The water-soluble natural Product samples to be detected can use multi-solvents dissolution, generally preferable using dissolubility, volatile
Solvent, preferably, the solvent is one of ethyl alcohol, methylene chloride, acetone, methanol or ethyl acetate etc. or a variety of；Into
One step is preferably one or both of ethyl alcohol or ethyl acetate.
It is unfolded step by step in solvent system after the water-soluble natural Product samples point sample to thin-layer chromatography, uses first
Be expanded to the distance of R1 on ethyl acetate/ethanol/water/acetic acid solvent plate, then dry, then with chloroform/ethyl acetate/
Formic acid solvent is expanded to the length of R2 again.
In the present invention, described R1, R2 be using initial point sample central point as starting point, be unfolded with solvent after front end
Line is the distance that terminal indicates.Preferably, the R1 is 15~25 millimeters；The R2 is 35~45 millimeters.Certainly, for spy
Fixed expansion system can also be controlled by the expansion effect that system is unfolded to two kinds in control duration of run.Preferably,
The R1 is 20 millimeters；The R2 is 40 millimeters.
In first solvent volume ratio of each solvent be ethyl acetate/ethanol/water/acetic acid=5:2.5~3/0.1~
0.2/0.1~0.15；Further preferred volume ratio are as follows: ethyl acetate/ethanol/water/acetic acid=5/2.85/0.15/0.12；Institute
The volume ratio for stating each solvent in the second solvent is chloroform/ethyl acetate/formic acid=1/0.8~1.2/0.01~0.05；
Further preferred volume ratio is chloroform/ethyl acetate/formic acid=1/1/0.03.
After the water-soluble natural Product samples are unfolded on thin-layer chromatography, carried out with P-methoxybenzal-dehyde color developing agent
Colour developing, developing time 0.1-10 minutes, 150-250 DEG C of colour temp.
The group of the coloring agent system become P-methoxybenzal-dehyde/acetic acid/ethyl alcohol/sulfuric acid volume ratio be 9.1~
9.5/3.5~4/300~350/12.5.Become as further preferred, described P-methoxybenzal-dehyde color developing agent group to first
Oxygroup benzaldehyde/acetic acid/ethyl alcohol/sulfuric acid (9.2/3.75/338/12.5, v/v/v/v).
In the present invention, the water-soluble natural product includes one of flavonoids, flavonoid glycoside, anthocyanin, saponin(e or more
Kind；As further preferred, the water-soluble natural product includes catechin, Quercetin, tea saponin, Cyanidin glucose
One of glycosides, oleanolic acid, chlorination Cyanidin, rutin are a variety of.
After the water-soluble natural Product samples develop the color on thin-layer chromatography, electronization is carried out using camera or scanner
Image Acquisition.
As shown in Figure 1, camera can be used or scanner carries out electronic Image Acquisition, complete water-soluble after the completion of colour developing
The electronization of Natural Product Samples image.After the water-soluble natural Product samples image electronic, suitable image is selected
Processing software, such as: ImageJ carries out image optimization processing.Since quantitative analysis is based on gray value, therefore first with software by picture
Switch to gray scale picture.
It includes denoising that the water-soluble natural Product samples image optimization, which is handled, smooth.To above-mentioned gray scale picture into
Row removes salt-pepper noise, corrects, and sharpens edge, smoothly waits sequence of operations, makes picture more suitable for quantitative analysis.
After the water-soluble natural Product samples image optimization processing, background removal is carried out.Suitable algorithm is selected, it will
Jamming pattern removes in image to be analyzed.
After the water-soluble natural Product samples image background removal, integrate quantitatively, obtain quantitative result.
For carrying out image procossing using ImageJ software, process are as follows:
After the water-soluble natural Product samples develop the color on thin-layer chromatography, electronics is carried out using HP M1005 scanner
Change Image Acquisition, parameters setting are as follows: million kinds of color outputs, resolution ratio 300dpi, brightness and contrast's default value, picture
It is saved with tiff format.
After the water-soluble natural Product samples image electronic, image optimization processing is carried out using ImageJ software.
Firstly, quantitative analysis is based on gray value, therefore start ImageJ software, first selects " Image "-" Type "-" 8-
Picture is converted into gray scale picture by bit ", then selects " Image "-" Adjust "-" Brightness/Contrast... ",
" Auto " is clicked in the dialog box of pop-up, picture contrast and lightness is adjusted, keeps area visualization degree to be analyzed higher.
Secondly, carrying out noise reduction and smoothly equal image optimizations processing to gray level image, first " Process "-" Noise "-is selected
" Despeckle ", by a median filter by each pixel in image replace with the intermediate value of pixel in its 3*3 neighborhood with
The salt-pepper noise in image is removed, deviation is more than to set by reselection " Process "-" Noise "-" RemoveOutliers... "
The pixel for determining threshold value replaces with the median of surrounding pixel to correct image；Then reselection " Process "-" Filters "-
" UnsharpMask... " extracts blurry versions to sharpen and enhance image border；Finally select " Process "-" Smooth "
Smooth whole image is so that integral baseline is more steady.
Then, after water-soluble natural Product samples image optimization processing, background is carried out using ImageJ software and is gone
It removes.It selects " Process "-" SubtractBackground... ", based on " Rollingball " algorithm in ImageJ, is popping up
Dialog box in give suitable ball radius, and choose " Lightbackground " and " Slidingparaboloid ", will
Continuous background in image removes.
After the water-soluble natural Product samples image background removal, integrate quantitatively using ImageJ software, obtain
To quantitative result." RectangularSelectionTool " selection target region is clicked, " Ctrl+1 " is clicked and determines band, point
It hits " Ctrl+3 " to be integrated, be connected peak both ends with " StraightLineSelectionTool " after forming closed figure, used
" wandtool " determines peak area, completes quantitative analysis.
The common chromatographic sheet of various specifications can be used in the present invention, preferably, standard curve with it is actually detected
Chromatographic sheet used is identical.
The present invention carry out quantitative analysis when, can construct in advance integrated value (for ImageJ be peak area) with sample size it
Between standard curve then after obtaining integrated value, the content of sample can be directly obtained, i.e. realization sample to be tested is determined
Quantization.
A kind of method of specific production standard curve is as follows:
(i) setting concentration (for example can be 1.0,2.0,3.0mg/L, can be that multiple groups are arranged in parallel) setting volume is taken
The standard sample of (can be 0.5 microlitre), carries out point sample on chromatographic sheet；
(ii) thin-layer chromatography is carried out to the chromatographic sheet after point sample using the first solvent and the second solvent to open up step by step
It opens；First solvent is ethyl acetate/ethanol/water/acetic acid composition expansion system, and second solvent is three chloromethanes
Alkane/ethyl acetate/formic acid composition expansion system；
(iii) it is developed the color using coloring agent system to the chromatographic sheet after expansion；
(iv) Image Acquisition is carried out to the chromatographic sheet after colour developing, obtains integrated value；
(v) according to step (i)~(iv) method, the corresponding integrated value of multiple groups standard sample is obtained, makes sample concentration
Standard curve between integrated value.
First solvent and the second solvent development distance can refer to the above-mentioned explanation to detection method, for example use first
Be expanded to the distance of R1 on ethyl acetate/ethanol/water/acetic acid solvent plate, then dry, then with chloroform/ethyl acetate/
Formic acid solvent is expanded to the length of R2 again.The R1 is 15~25 millimeters；The R2 is 35~45 millimeters.Certainly, for
Specific expansion system can also be controlled by the expansion effect that system is unfolded to two kinds in control duration of run.As excellent
The solvent system of choosing, actually detected use is identical as the expansion system that production standard curve uses.
Preferably, development distance is identical as actually detected development distance in the process, the point of use when building standard curve
Batten part, color condition and Image Acquisition and treatment conditions with it is consistent in detection process.
The present invention can be used for the detection of single component sample, can be used for the detection of mixed composition sample, be used for
When the detection of mixed composition sample, the Image Acquisition to each component sample spot is realized respectively using image acquisition units, then
Calculate separately the concentration value of sample.Certainly, using the present invention, we can also once realize the detection of multiple samples, when detection,
Multiple samples can be subjected to point sample on the same chromatographic sheet, then carry out Image Acquisition and calculating respectively.Such as Fig. 1 institute
Show, for six concentration, successively raised sample is detected using method of the invention, i.e., sample is distinguished point sample to same
Point sample position on chromatographic sheet, as long as distance can be avoided interference between each other；Then it is unfolded, develops the color, so
It is scanned afterwards using computer scanning instrument, reads the corresponding image of each sample respectively, integrated, finally obtain corresponding sample
The concentration value of product, testing result is suitable with individually detection, almost without interference effect.
The present invention has the advantages that relative to existing detection method
(1) water-soluble natural product thin layer quantitative image recognition detection method of the invention is easy to operate, does not need dedicated
Instrument.
(2) method of the invention can detect a variety of water-soluble natural products using same expansion system, fixed
Amount property is good, easy to operate, and accuracy is high.
(3) method test speed of the invention is fast, can be measured simultaneously with multiple samples；It can also be to containing multiple components
Sample is detected, practical.
Detailed description of the invention
Fig. 1 is for six concentration procedure chart that successively raised sample uses method of the invention to be detected.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Color developing agent used in the examples is P-methoxybenzal-dehyde coloring agent, consisting of P-methoxybenzal-dehyde/second
Acid/ethyl alcohol/sulfuric acid (9.2/3.75/338/12.5, v/v/v/v).
Standard curve is made first, the method is as follows:
(i) standard sample for taking setting concentration, it is enterprising in chromatographic sheet (using the same chromatographic sheet of embodiment 1)
Row point sample；In the present embodiment, using 0.5,1.0,2.0,3.0,4.0mg/L five group of standard sample, (sample and each embodiment are needed
The sample to be detected is identical), solvent is ethyl alcohol, and point sample volume is 0.5 microlitre；
(ii) after dry, thin-layer chromatography is carried out to the chromatographic sheet after point sample using the first solvent and the second solvent
It is unfolded step by step；
With 20 millimeters of expansion on ethyl acetate/ethanol/water/acetic acid (5/2.85/0.15/0.12, v/v/v/v) solvent plate
Distance, then dry, then be expanded to 40 again with chloroform/ethyl acetate/formic acid (1/1/0.03, v/v/v) solvent
The length of millimeter；
(iii) it is developed the color using P-methoxybenzal-dehyde coloring agent to the chromatographic sheet after expansion: 250 DEG C of warm tables
Heating develops the color for 15 seconds；
(iv) it is scanned after thin layer colour developing with computer scanner, resolution ratio is set as 300dpi.Scan obtained figure
Picture carries out denoising, smoothing processing with ImageJ, then removes background, finally carries out quantitative integration, respectively obtains 0.5,1.0,
2.0,3.0,4.0mg/L five groups of corresponding integrated values of standard sample；
(v) standard curve between sample concentration and integrated value is made.
For the accuracy of detection, for every group of standard sample, Parallel testing three times, takes the average value of integrated value right for its
The integrated value answered.
For the sample of different component, it is required to carry out the drafting of standard curve according to the method described above.
After sample colour developing during production standard curve and in embodiment, subsequent image treatment process are as follows:
Electronic Image Acquisition, parameters setting are carried out to the chromatographic sheet after colour developing using HP M1005 scanner
Are as follows: the output of hundred million colors, resolution ratio 300dpi, brightness and contrast's default value, picture are saved with tiff format.
Image optimization processing is carried out using ImageJ software.Firstly, quantitative analysis is based on gray value, therefore it is soft to start ImageJ
Part first selects " Image "-" Type "-" 8-bit ", picture is converted into gray scale picture, then selects " Image "-
" Adjust "-" Brightness/Contrast... ", in the dialog box of pop-up click " Auto ", adjustment picture contrast and
Lightness keeps area visualization degree to be analyzed higher.
Secondly, carrying out noise reduction and smooth etc. reason to gray level image, first " Process "-" Noise "-is selected
" Despeckle ", by a median filter by each pixel in image replace with the intermediate value of pixel in its 3*3 neighborhood with
The salt-pepper noise in image is removed, deviation is more than to set by reselection " Process "-" Noise "-" RemoveOutliers... "
The pixel for determining threshold value replaces with the median of surrounding pixel to correct image；Then reselection " Process "-" Filters "-
" Unsharp Mask... " extracts blurry versions to sharpen and enhance image border；Finally select " Process "-" Smooth "
Smooth whole image is so that integral baseline is more steady.
Then, background removal is carried out using ImageJ software.It selects " Process "-
" SubtractBackground... " is given in the dialog box of pop-up and is closed based on " Rollingball " algorithm in ImageJ
Suitable ball radius, and " Lightbackground " and " Slidingparaboloid " are chosen, by the continuous background in image
It removes.
After image background removal, integrate quantitatively using ImageJ software, obtain quantitative result.It clicks
" RectangularSelectionTool " selection target region clicks " Ctrl+1 " and determines band, clicks " Ctrl+3 " and carries out
Integral is connected peak both ends with " StraightLine SelectionTool " after forming closed figure, true with " wandtool "
Determine peak area, obtains corresponding integrated value.
Embodiment 1
By (+)-catechin ethanol solution, with 0.5 microlitre of capillary point sample to thin-layer chromatography after solvent volatilizes, use acetic acid
20 millimeters of distance is unfolded on ethyl ester/ethanol/water/acetic acid (5/2.85/0.15/0.12, v/v/v/v) solvent plate, then dries in the air
It does, then is expanded to 40 millimeters of length again with chloroform/ethyl acetate/formic acid (1/1/0.03, v/v/v) solvent.So
After take out after solvent volatilizes with P-methoxybenzal-dehyde staining reagent, be placed into 250 DEG C of warm tables and heat 15 seconds and shown
Color.It is scanned after thin layer colour developing with computer scanner, resolution ratio is set as 300dpi.Scan obtained image ImageJ
Denoising, smoothing processing are carried out, background is then removed, finally carries out quantitative integration, integrated value compared with standard value by obtaining
Measurement result.Actual value is (+)-catechin solution of 1.20mg/mL, measured value 1.22mg/mL, error 2%.
The chromatographic sheet of use is purchased from market, and specification (silica gel material, coating layer thickness: 0.2-0.25 millimeters, silica white
Granularity: 10-40 microns).
Embodiment 2
By Quercetin ethanol solution, with 0.5 microlitre of capillary point sample to thin-layer chromatography after solvent volatilizes, with acetic acid second
20 millimeters of distance is unfolded on ester/ethanol/water/acetic acid (5/2.85/0.15/0.12, v/v/v/v) solvent plate, then dries,
It is expanded to 40 millimeters of length again with chloroform/ethyl acetate/formic acid (1/1/0.03, v/v/v) solvent again.Then it takes
P-methoxybenzal-dehyde staining reagent is used after solvent volatilizes out, 250 DEG C of warm tables is placed into and heats 15 seconds and develop the color.It is thin
It is scanned after layer colour developing with computer scanner, resolution ratio is set as 300dpi.Obtained image is scanned to be carried out with ImageJ
Then denoising, smoothing processing remove background, finally carry out quantitative integration, integrated value compared with standard value by being measured
As a result.Actual value is 1.51mg/mL by Quercetin ethanol solution, measured value 1.53mg/mL, error 1.5%.
Embodiment 3
By tea saponin ethanol solution, with 0.5 microlitre of capillary point sample to thin-layer chromatography after solvent volatilizes, with acetic acid second
20 millimeters of distance is unfolded on ester/ethanol/water/acetic acid (5/2.85/0.15/0.12, v/v/v/v) solvent plate, then dries,
It is expanded to 40 millimeters of length again with chloroform/ethyl acetate/formic acid (1/1/0.03, v/v/v) solvent again.Then it takes
P-methoxybenzal-dehyde staining reagent is used after solvent volatilizes out, 250 DEG C of warm tables is placed into and heats 15 seconds and develop the color.It is thin
It is scanned after layer colour developing with computer scanner, resolution ratio is set as 300dpi.Obtained image is scanned to be carried out with ImageJ
Then denoising, smoothing processing remove background, finally carry out quantitative integration, integrated value compared with standard value by being measured
As a result.Actual value is 2.5mg/mL by tea saponin ethanol solution, measured value 2.62mg/mL, error 5%.
Embodiment 4
By C-3-G ethanol solution, with 0.5 microlitre of capillary point sample to thin-layer chromatography after solvent volatilizes,
With 20 millimeters of distance is unfolded on ethyl acetate/ethanol/water/acetic acid (5/2.85/0.15/0.12, v/v/v/v) solvent plate,
Then it dries, then is expanded to 40 millimeters of length again with chloroform/ethyl acetate/formic acid (1/1/0.03, v/v/v) solvent
Degree.Then take out after solvent volatilizes with P-methoxybenzal-dehyde staining reagent, be placed into 250 DEG C of warm tables heat 15 seconds into
Row colour developing.It is scanned after thin layer colour developing with computer scanner, resolution ratio is set as 300dpi.Obtained image is scanned to use
ImageJ carries out denoising, smoothing processing, then removes background, finally carries out quantitative integration, integrated value by with standard value ratio
Relatively obtain measurement result.Actual value is the C-3-G solution of 1.50mg/mL, measured value 1.53mg/mL, error
It is 2%.
Embodiment 5
Oleanolic acid, Quercetin, (+)-catechin, chlorination Cyanidin, rutin ethanol solution (actual content difference
Are as follows: oleanolic acid: 1.21mg/mL；Quercetin: 1.31mg/mL；(+)-catechin: 1.17mg/mL；Chlorination Cyanidin:
2.54mg/mL；Rutin: 1.12mg/mL), with 0.5 microlitre of capillary point sample to thin-layer chromatography after solvent volatilizes, with acetic acid second
20 millimeters of distance is unfolded on ester/ethanol/water/acetic acid (5/2.85/0.15/0.12, v/v/v/v) solvent plate, then dries,
It is expanded to 40 millimeters of length again with chloroform/ethyl acetate/formic acid (1/1/0.03, v/v/v) solvent again.Then it takes
P-methoxybenzal-dehyde staining reagent is used after solvent volatilizes out, 250 DEG C of warm tables is placed into and heats 15 seconds and develop the color.It is thin
It is scanned after layer colour developing with computer scanner, resolution ratio is set as 300dpi.Obtained image is scanned to be carried out with ImageJ
Then denoising, smoothing processing remove background, finally carry out quantitative integration, integrated value compared with standard value by being measured
As a result, obtaining content value is oleanolic acid: 1.17mg/mL；Quercetin: 1.27mg/mL；(+)-catechin: 1.15mg/mL；
Chlorination Cyanidin: 2.46mg/mL；Rutin: 1.09mg/mL.Below error 3%.
Claims (10)
1. a kind of water-soluble natural product thin layer quantitative image recognition detection method characterized by comprising
(1) sample to be tested containing water-soluble natural product carries out point sample on chromatographic sheet；
(2) thin-layer chromatography is carried out to the chromatographic sheet after point sample using the first solvent and the second solvent to be unfolded step by step；Institute
Stating the first solvent is ethyl acetate/ethanol/water/acetic acid composition expansion system, and second solvent is chloroform/second
Acetoacetic ester/formic acid composition expansion system；
(3) it is developed the color using coloring agent system to the chromatographic sheet after expansion；
(4) Image Acquisition is carried out to the chromatographic sheet after colour developing, according to the pass between image and water-soluble natural product assay
System, obtains the content of water-soluble natural product.
2. water-soluble natural product thin layer quantitative image recognition detection method according to claim 1, which is characterized in that institute
State each solvent in the first solvent volume ratio be ethyl acetate/ethanol/water/acetic acid=5/2.5~3/0.1~0.2/0.1~
0.15；The volume ratio of each solvent is chloroform/ethyl acetate/formic acid=1/0.8~1.2/0.01 in second solvent
~0.05.
3. water-soluble natural product thin layer quantitative image recognition detection method according to claim 1, which is characterized in that step
Suddenly in (2), it is expanded to the distance of R1 first with the first solvent, then dries, then be expanded to R2 again with the second solvent
Distance, the R1 are 15~25 millimeters；The R2 is 35~45 millimeters.
4. water-soluble natural product thin layer quantitative image recognition detection method according to claim 1, which is characterized in that institute
Stating coloring agent system is P-methoxybenzal-dehyde color developing agent.
5. water-soluble natural product thin layer quantitative image recognition detection method according to claim 4, which is characterized in that institute
State the composition volume of coloring agent system are as follows: P-methoxybenzal-dehyde/acetic acid/ethyl alcohol/sulfuric acid=9.1~9.5/3.5~4/300~
350/12.5。
6. water-soluble natural product thin layer quantitative image recognition detection method according to claim 1, which is characterized in that institute
The concentration for stating water-soluble natural product in sample to be tested is 0.01-10mg/mL, and point sample amount is 0.1-10 microlitres.
7. water-soluble natural product thin layer quantitative image recognition detection method according to claim 1, which is characterized in that adopt
Electronic Image Acquisition is carried out with camera or scanner.
8. water-soluble natural product thin layer quantitative image recognition detection method according to claim 1, which is characterized in that right
The image of acquisition is handled using ImageJ, and finally carries out integrating quantitatively, utilizes the pass between integrated value and sample size
System, obtains the concentration of the sample to be tested containing water-soluble natural product.
9. water-soluble natural product thin layer quantitative image recognition detection method according to claim 1, which is characterized in that pre-
First construct the standard curve between sample size and integrated value, it is bent according to obtained integrated value and the standard when detection
Line obtains the concentration of the sample to be tested containing water-soluble natural product.
10. water-soluble natural product thin layer quantitative image recognition detection method according to claim 1, which is characterized in that
The water-soluble natural product is one of flavonoids, flavonoid glycoside, anthocyanin, saponin(e or a variety of.
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