CN103487412B - Method for constructing rapid hepatotoxicity predication model of phenolic compound - Google Patents
Method for constructing rapid hepatotoxicity predication model of phenolic compound Download PDFInfo
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Abstract
The invention belongs to the technical field of rapid toxic substance detection, and particularly relates to a method for constructing a rapid hepatotoxicity predication model of a phenolic compound. According to the method, an HepG2/C3A cell with stable inheritance and high-expression cytochrome P450 is taken as a research object; 6 types of natural common phenolic compounds are selected and act on the cell respectively, and indexes such as cell CYPIA mitochondrial depolarization, ROS production rate and the like are tested respectively; and statistical analysis is combined, and finally, the prediction model is established. The method is simple and rapid and is used for predicating the hepatotoxicity of a drug, the result is stable, the accuracy is improved, and the method has a potential popularization and application prospect.
Description
Technical field
The invention belongs to technical field of rapid toxic substance detection, be specifically related to a kind of construction method of rapid hepatotoxicity predication model of phenolic compound.
Background technology
In daily life, many phenolic compounds of people's picked-up have the harm causing hepatic injury, but after hepatic injury, have many response indexs, such as hepatic injury can cause cell mitochondrial current potential depolarizing influences normal function, the excessive generation causing ROS free radical and DNA damage, Cytochrome P450 abnormal expression etc., but these indexs are incomplete.Therefore, a kind of index of simple use judges it is inaccurate.
The present invention is caused hepatic injury response index to phenolic compound screened by early stage, in conjunction with clone genetic stability characteristic, and carries out chemometrics method to index and carries out cluster analysis and discriminating, constructed simple and quick Forecasting Methodology.
Summary of the invention
For overcoming the deficiencies in the prior art, the invention provides a kind of construction method of rapid hepatotoxicity predication model of phenolic compound.The method is used for the hepatotoxic prediction of phenolic compound, and the result obtained is stable, fast, accuracy improves, and has potential application prospect.
The inventive method relates to the foundation of detection system, the Chemical Measurement visual analyzing of multi-dimensional complicated data target and modeling.This modeling method integrates multiple cell index all sidedly, in conjunction with the complex data projective clustering method in Chemical Measurement, can carry out comprehensive assessment to noxious material.
The construction method of a kind of rapid hepatotoxicity predication model of phenolic compound provided by the invention, there is genetic stability and Cytochrome P450 has the HepG2/C3A clone of high expressed as research object by choosing, choose more than 6 kinds phenolic compounds, it is made to act on cell respectively, the indexs such as cell CYP1A2 mitochondrial depolarization, ROS production rate are tested respectively, in conjunction with statistical analysis, finally establish forecast model.Concrete steps comprise:
(1) foundation of detection system
Select HepG2/C3A clone, cultivate 24-36h after bed board, now plating cells density is 1000 ~ 20000/ holes, and cell algebraically was 4 ~ 8 generations; It is under the effect of phenolic compound, through step 1., step 2. with step 3. duplicate detection, obtain the data set of the mitochondrial depolarization of phenolic compound, ROS production rate, CYP1A enzyme activity and CYP2B3A enzyme activity 4 indexs respectively;
1. mitochondrial depolarization test: cell stimulates after 2-3h through phenolic compound, install probe Rhodamine 123 additional, acting in conjunction about 30-40min, by liquid sucking-off in orifice plate, HBSS cleaning after, add phenolic compound 24-36h, after in microplate reader excitation wavelength 490nm, absorbing wavelength 535nm detects, and obtains mitochondrial depolarization index;
2. ROS production rate test: early stage install probe DCFHDA additional, sucking-off after 30-40min, HBSS cleaning after in cell, add phenolic compound effect 24-36h, after in microplate reader excitation wavelength 485nm, absorbing wavelength 525nm detects, and obtains ROS production rate index;
3. cytochrome p450 tests: 7-ethoxyresorufin and 7-benzyloxy resorufin are respectively as substrate, after phenolic compound function cells 24-36h, sucking-off phenolic compound is with after HBSS cleaning, add working fluid, then above two kinds of substrates are added respectively, be placed in microplate reader excitation wavelength 544nm, absorbing wavelength 590nm detects, and obtains CYP1A, CYP2B3A enzyme activity index respectively;
(2) the Chemical Measurement visual analyzing of multi-dimensional complicated data target
Adopt principal component analysis (PCA) (Principal Component Analysis, PCA) algorithm, first carries out Chemical Measurement visual analyzing to the data set of 4 indexs obtained in step (1), obtains score, carry out classification to pharmaceutical samples by this to judge, select major component; Again the load of major component is analyzed, for setting up applicable parameterized model;
(3) foundation of comprehensive analysis indexes
According to the result of Chemical Measurement visual analyzing, the load of the first two major component that the contribution rate obtained with principal component analysis (PCA) is maximum is parametric model, the standard items to modeling in conjunction with the load of major component carry out comprehensive cluster situation analysis, the sample be defined in certain major component load range is have similar toxicity and toxicology property with these type of close standard items, set up comprehensive analysis indexes in this approach, realize the foundation of forecast model.
In the present invention, when step (1) sets up detection system, choose 6 kinds of phenolic compounds and act on HepG2/C3A clone, it is made up of gallic acid, nordihydroguaiaretic acid, green tea, Quercetin and caffeic acid and Epigallo-catechin gallate (EGCG).
In the present invention, bottom the middle cytochrome p450 of step (1) tests and adopts black, opaque plate carries out plating cells, and mitochondrial depolarization is tested and bottom ROS production rate test employing black, transparent panel carries out plating cells.
PCA algorithm is adopted to carry out Chemical Measurement visual analyzing in the present invention.Traditional biological evaluation index mapping mode mostly is histogram.Its effect in discussion single argument evaluation index is intuitively effective, but when evaluation index becomes many, chart number increases, and thus analyzes numerous and diverse, is difficult to fast and effeciently analyze.And PCA is a kind of projecting method, its by multivariate data through mathematic(al) manipulation in a sub spaces, the deviation (variance) of each independent data sample room can be embodied as far as possible.The result of projection is called score (score), can show by a two dimension or three-dimensional projection.It reflects the size of each sample room degree associated with each other, can judge the classification of sample by this.The direction of projection is called load (loading), can show, it reflects the significance level of each variable of measurement with line graph or histogram.Principal component analytical method utilizes linear combination, i.e. one group of major component, calculates the score a sample set data projection to low-dimensional, and maintains information useful in sample data, can reflect the character etc. of sample to each sample.
The response change that the index that the present invention chooses is common after can containing hepatic injury, HepG2/C3A clone has stable response, and index is reliably with accurately.
Accompanying drawing explanation
Fig. 1 is R123 sample determination result.
Fig. 2 is ROS sample determination result.
Fig. 3 is CYP1A sample determination result.
Fig. 4 is CYP2B/3A sample determination result.
Fig. 5 is the score vector projection of principal component analysis (PCA).
Fig. 6 is major component load (loading) figure.
Fig. 7 is the score vector projection of principal component analysis (PCA), and tannic acid test set represents with X in the drawings.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in more detail.
Embodiment 1
1. select gallic acid, nordihydroguaiaretic acid (NDCG), green tea, Quercetin, caffeic acid, 6 kinds of common phenolic compounds such as Epigallo-catechin gallate (EGCG) (EGCG), the concentration of monomer is 10mg/ml, and extract concentrations is 100mg/ml.Preparation mother liquor, dissolves with dimethyl sulfoxide (DMSO) (DMSO), guarantees that DMSO concentration controls below 0.5%.
2. plating cells density is in 20000/ hole, 200 μ l, after 24 hours, after supernatant sucking-off, add 200 μ l medicines, adding whole bulking value concentration after effect 2.5h is 5 μ g/ml Rhodamine 123s, by supernatant sucking-off after 30min, after HBSS cleaning, then add 200 μ l medicines, detect in microplate reader excitation wavelength 490nm, absorbing wavelength 535nm after 24 hours.Testing result as shown in Figure 1.
3.ROS production rate is tested: plating cells concentration is in 20000/ hole, 200 μ l, after 24 hours, after supernatant sucking-off, add 200 μ l, 5 μm of DCFHDA probes, by after supernatant sucking-off after 30min, with HBSS cleaning, then add 200 μ l medicines, 24h is placed on microplate reader excitation wavelength 485nm, and absorbing wavelength 525nm detects.Testing result as shown in Figure 2.
4.CYP1A, CYP2B3A enzyme activity is tested: plating cells concentration is in 20000/ hole, 200 μ l, after 24 hours, after supernatant sucking-off, add the sample that 200 μ l prepare, by supernatant sucking-off after 24h, 50 μ l working fluids (potpourri of salicylamide and bicoumarin) are added with after HBSS cleaning, after add enzyme reaction substrate 7-ethoxyresorufin and the 7-benzyloxy resorufin that 50 μ l shift to an earlier date preheating, be placed on microplate reader excitation wavelength 544nm, absorbing wavelength 590nm detect.Testing result as shown in Figure 3, Figure 4.
5. the chemometrics application of sample: all samples all carries out six replications.Can draw a preliminary conclusion from data above, different indexs lays particular emphasis on measures toxicity to the effect of cell different aspect.Thus, the result that draws of the system of many evaluations is not quite identical.Such as, the hepatotoxicity wind agitation of NDCG has all showed obvious toxicity in except other 3 indexs of Rhodamine 123, and green tea at 3 index Poisonings clearly, except ROS index is different.Equally, other sample responds difference in different indexs.Therefore, comprehensive assessment is done to toxicity and need more other mathematical method and means.Six replications through blank sample and six samples establish 42 data sets recorded.Data set input MATLAB R2013a software processes.Fig. 5 sees in the score vector projection of its principal component analysis (PCA).
In Fig. 5, the unit dimensionless of horizontal ordinate and ordinate.The score (score) of major component 1 and 2 is respectively in x-axis and y-axis.A is that 0.5%DMSO solution is blank, and B is gallic acid, and C is NDCG, D is green tea, and E is Quercetin, and F is caffeic acid, and G is EGCG.Every class sample is with different color markings.As can be seen from the figure, sample collection is obvious several classification.NDCG and green tea are larger with the distance of blank sample separately, and distance between them is also larger.Judge in conjunction with single index result above, both there is obvious hepatotoxicity wind agitation, and both toxicity mechanisms are also different.Also there is distance in tannic acid and gallic acid and blank solution, can be inferred as middle hypotoxicity material.Quercetin, caffeic acid, the distance between HGCG tri-material and blank sample is very near, close to nontoxicity.
Fig. 6 denotes the load (loading) of major component 1 and 2.Load is in principal component analysis (PCA), and each major component is for the weight of variable.It represents model and variable relation important indicator, can reflect the relation of each variable of data and the size of data field calibration.Its absolute value is larger, then show that the importance of this Testing index is larger.In Fig. 6, ROS is very large for the contribution of major component 1, and R123 then contributes very little for major component 1.For major component 2, situation is just in time contrary.CYP1A and CYP2B/3A belongs to enzyme activity test, and its importance is also roughly convergent in two major components.This phenomenon reflects green tea and NDCG has different responses to ROS and R123 two indices, meets univariate analysis result above, also demonstrates the toxicity information utilizing principal component analysis (PCA) comprehensively can excavate various index to comprise.
6. the foundation of comprehensive analysis indexes and checking: entered above-mentioned visual analyzing step, and using major component 1 and 2 as parametric model, proposed the comprehensive analysis indexes of two indices as toxicant:
P1=-0.029577236*R123+0.925580291*ROS+0.298203392
*CYP1A+0.231302939*CYP2B/3A
P2=-0.764237679*R123-0.243863606*ROS+0.266833164*CYP1A+0.534108018*CYP2B/3A
Described index R123, ROS, CYP1A, CYP2B/3A have to pass through centralization process, the mean value of this index of data centralization when namely initial value deducts modeling.
Tannic acid is selected to carry out comprehensive evaluation as the new sample sets index set up.The step of four cell toxicity index determinings is consistent with above-mentioned steps, and the index obtained is as following table:
Table 1: the raji cell assay Raji result of tannic acid
| R123 | 190.6154 | 188.3313 | 181.4712 | 193.1771 | 181.5822 | 185.1035 |
| ROS | 240.3302 | 232.2174 | 231.0536 | 230.4465 | 213.9847 | 224.8299 |
| CYP1A | 18.98994 | 14.85191 | 10.44295 | 6.443319 | 8.149972 | 7.302705 |
| CYP2B/3A | 23.91249 | 17.65922 | 13.5079 | 9.210743 | 10.86263 | 9.434631 |
| P1 | -11.17475 | -21.29662 | -24.44588 | -27.540684 | -41.543440 | -32.192446 |
| P2 | -46.92352 | -47.64360 | -45.51075 | -57.671175 | -43.457804 | -49.782449 |
The two indices calculated can be made on major component figure, as shown in Figure 7, and carries out conclusion supposition toxicity according to existing cluster.By analysis, tannic acid liver poison toxicology property and toxic degree and gallic acid the most close.This is consistent with the toxicity rule summed up in the past.By above example, we can draw the gordian technique incorporated in cell biology toxicity and Chemical Measurement of this patent innovation, for setting up a kind of science, objective and clearly appraisement system established working foundation.
Although the specific algorithm of preferred embodiment and demonstration discloses as above by the present invention; but itself and be not used to limit content of the present invention; anyly be familiar with cell toxicity evaluation system and statistics modeling algorithm person; not departing from main spirits of the present invention and context; when doing various change and retouching; comprise change, interpolation, delete evaluation index, change chemometrics algorithm, the protection domain therefore invented should be as the criterion with the basic right claimed range applied for a patent.
Claims (7)
1. the construction method of a rapid hepatotoxicity predication model of phenolic compound, it is characterized in that, choose more than 6 kinds phenolic compounds, it is made to act on HepG2/C3A cell respectively, cell CYP1A2 mitochondrial depolarization, ROS production rate, CYP1A enzyme activity and CYP2B/3A enzyme activity index are tested respectively, in conjunction with statistical analysis, finally establish forecast model; Concrete steps comprise:
(1) foundation of detection system
Select HepG2/C3A clone, cultivate 24-36h after bed board, now plating cells density is 10000 ~ 20000/ holes, and cell algebraically was 4 ~ 8 generations; It is under the effect of phenolic compound, through step 1., step 2. with step 3. duplicate detection, obtain the data set of the mitochondrial depolarization of phenolic compound, ROS production rate, CYP1A enzyme activity and CYP2B/3A enzyme activity 4 indexs respectively;
1. mitochondrial depolarization test: cell stimulates after 2-3h through phenolic compound, install probe Rhodamine 123 additional, acting in conjunction about 30-40min, by liquid sucking-off in orifice plate, HBSS cleaning after, add phenolic compound 24-36h, after in microplate reader excitation wavelength 490nm, absorbing wavelength 535nm detects, and obtains mitochondrial depolarization index;
2. ROS production rate test: early stage install probe DCFHDA additional, sucking-off after 30-40min, HBSS cleaning after in cell, add phenolic compound effect 24-36h, after in microplate reader excitation wavelength 485nm, absorbing wavelength 525nm detects, and obtains ROS production rate index;
3. cytochrome p450 tests: 7-ethoxyresorufin and 7-benzyloxy resorufin are respectively as substrate, after phenolic compound function cells 24-36h, sucking-off phenolic compound is with after HBSS cleaning, add working fluid, then above two kinds of substrates are added respectively, be placed in microplate reader excitation wavelength 544nm, absorbing wavelength 590nm detects, and obtains CYP1A, CYP2B/3A enzyme activity index respectively;
(2) the Chemical Measurement visual analyzing of multi-dimensional complicated data target
Adopt the algorithm of principal component analysis (PCA), first Chemical Measurement visual analyzing is carried out to the data set of 4 indexs obtained in step (1), obtain score, by this classification is carried out to pharmaceutical samples and judge, select major component; Again the load of major component is analyzed, for setting up applicable parameterized model;
(3) foundation of comprehensive analysis indexes
According to the result of Chemical Measurement visual analyzing in step (2), the load of the first two major component that the contribution rate obtained with principal component analysis (PCA) is maximum is parametric model, in conjunction with the load of major component, comprehensive cluster situation analysis is carried out to the standard items of modeling, be defined in sample in certain major component load range and these type of close standard items have similar toxicity and toxicology property, set up comprehensive analysis indexes in this approach, realize the foundation of forecast model.
2. construction method according to claim 1, is characterized in that: described phenolic compound is 6 kinds, and it is made up of gallic acid, nordihydroguaiaretic acid, green tea, Quercetin and caffeic acid and Epigallo-catechin gallate (EGCG).
3. construction method according to claim 1, it is characterized in that: bottom the middle cytochrome p450 of step (1) tests and adopts black, opaque plate carries out plating cells, mitochondrial depolarization is tested and bottom ROS production rate test employing black, transparent panel carries out plating cells.
4. construction method according to claim 1, is characterized in that: working fluid described in step (1) is the potpourri of salicylamide and bicoumarin.
5. construction method according to claim 1, is characterized in that: in step (1), and 4 indexs repeat 6 times and detect.
6. construction method according to claim 1, is characterized in that: mitochondrial depolarization index R123, and ROS, CYP1A, CYP2B/3A, through centralization process, are the mean value of this index of data centralization when initial value deducts modeling.
7. construction method according to claim 1, is characterized in that: in step (3), with green tea and nordihydroguaiaretic acid for parametric model, and the comprehensive analysis indexes using P1 and P2 two indices as toxicant, concrete formula is as follows:
P1＝-0.029577236*R123+0.925580291*ROS+0.298203392
*CYP1A+0.231302939*CYP2B/3A；
P2＝-0.764237679*R123-0.243863606*ROS+0.266833164*CYP1A+0.534108018*CYP2B/3A；
Wherein: R123 represents mitochondrial depolarization index; ROS represents ROS production rate index; CYP1A represents CYP1A enzyme activity index; CY P2B/3A represents P2B/3A enzyme activity index.
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