CN102335221A - Method for screening active component with function of hexokinase inhibition effects - Google Patents
Method for screening active component with function of hexokinase inhibition effects Download PDFInfo
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Abstract
The invention belongs to the field of drug analysis, relates to a method for screening an active component in a traditional Chinese medicine, and specifically, relates to a method for screening a traditional Chinese medicine active component with a function of hexokinase inhibition effects. Based on hexokinase catalytic reaction adopting adenosine triphosphate (ATP) as a substrate, the method can screen out a traditional Chinese medicine active component with a function of hexokinase inhibition effects through quantitative analysis processes on ATP and product ATP. The method is rapid, simple, economic and effective. Compared with a cell test method, the method reduces an extract use amount, saves biochemical reagents, and shortens a screening period. Therefore, the method has great importance for screening and research of traditional Chinese medicine active components. In the invention, all experiment parameters of an improved liquid chromatography-mass spectrometry (LC-MS) method have good reference values for mass spectrometry technology-based determination of other biomolecules with similar properties.
Description
Technical field
The invention belongs to the pharmaceutical analysis field, relate to the screening technique of active component in the Chinese crude drug, be specifically related to the screening technique of the inhibiting active ingredient of Chinese herbs of a kind of tool hexokinase.
Background technology
Malignant tumor is serious threat human life's a difficult diseases, is to be only second to cardiopathic second killer.At present, clinical drug treatment commonly used is a chemotherapy, yet regular meeting brings and serious adverse during chemotherapy.Therefore, from natural resources, seek and screening antineoplastic active component, develop the medicine of effective low toxicity, caused medical worker's extensive concern.But practice confirms, crude drug, and like Chinese crude drug, its composition complicacy very is for wherein active ingredient screening brings great challenge.Be necessary to seek a kind of quick, effective screening technique.
As everyone knows, drug effect be a series of biological effects through combining with target molecule to cause, this effect directly influences distribution, drainage, metabolism, activity and the toxic and side effects of medicine.Chinese medicine extract is mixed with the effect target molecule under certain condition, have interactional active component with target molecule reversible the combination takes place with it, form bioactive molecule-target molecule complex.Research shows, if obtain the information before and after active ingredient of Chinese herbs and the effect of many target spots, just can disclose and the bonded reactive compound of target molecule.For example select certain organized enzyme as target molecule, measure this target molecule through certain means and combine the amount of its substrate of back or product whether to change with Chinese medicine extract, can judge tentatively that then Chinese medicine extract has non-activity.
(hexokinase HK) is the enzyme that the catalysis hexose makes it phosphorylation, in the normal tissue expression amount seldom to hexokinase.It is first enzyme of glycolytic pathway, also is the rate-limiting enzyme of glycolytic pathway.Amount that hexokinase is expressed in tumor tissues and active increase; Make tumor tissues under the situation of deficiency of oxigen; Still can guarantee the enough energy; And glucolytic many intermediate products can be utilized by oncocyte, with synthetic protein, nucleic acid and lipid etc., thereby essential material base are provided for the growth of oncocyte itself and hypertrophy.Discover that tumor has high glycometabolic characteristics: about 60% ATP derives from glycolytic pathway in the tumor cell.Seeing that hexokinase is for the active significance of various tumor cells; Research worker is rationally inferred, if can search out effective hexokinase inhibitor, suppresses the activity of this enzyme; The glycolysis that has then suppressed tumor cell; Make tumor cell can not obtain enough 6-glucose 1-phosphate1-s, ATP can't convert ADP into and for the cell comings and goings energy is provided, and causes tumor cell to take place to transfer then and dies.
Summary of the invention
The purpose of this invention is to provide the active in-vitro screening method of a kind of easy, quick and economic Chinese crude drug.Be specifically related to the screening technique of the inhibiting active ingredient of Chinese herbs of a kind of tool hexokinase.The screening technique of the inhibiting activity extract of the auxiliary tool hexokinase of especially a kind of LC-MS method.
The inventive method is based in the glycolytic cycle; Hexokinase catalysis ATP is converted into the process of ADP; To treat that screening of medicaments or component add in this reaction system; With the LC-MS method ATP and ADP are carried out quantitative assay, observe the situation of change of ATP and ADP and treat with evaluation whether screening of medicaments or component have the activity of hexokinase inhibitor, and its activity is measured.
Particularly; The screening technique of the inhibiting active ingredient of Chinese herbs of a kind of tool hexokinase of the present invention is characterized in that, is the hexokinase catalytic reaction of substrate with ATP; Through quantitative analysis to ATP and product A DP; Filter out the inhibiting active component of tool hexokinase in the Chinese crude drug, it comprises step
(1) set up high performance liquid chromatogram-mass spectrometry combination method (LC-MS),
Through in mobile phase, adding the ammonium acetate of 5mM, realize detection and quantitative analysis to ATP and product A DP;
Among the present invention, for the minimum 5 μ g/mL that quantitatively are limited to of polar molecule ATP.
Among the present invention, mobile phase need be added the ammonium acetate of 5mM, is detector with the mass spectrum, and capillary voltage is set to 3000V, and the monitoring ion is respectively ATP:m/z 508, and ADP:m/z 428.
(2) based on LC-MS ATP is carried out quantitative analysis, measure with the hexokinase activity of ATP as substrate;
The Chinese medicine extract MBZA of the tool hexokinase inhibitor activity that (3) screening is obtained measures and calculates.
In one embodiment of the present of invention, selecting the Chinese crude drug Semen Momordicae for the screening object, is means with LC-MS; Measure the hexokinase activity of ATP as substrate; (MBZA, MBZB, MBZC and MBZD) carries out the screening of hexokinase inhibitor activity to four kinds of extracts of Semen Momordicae; Described Chinese crude drug Semen Momordicae has been used in the anti-tumor compound, and pharmacology confirms that it has mass dissipating and swelling eliminating, and counteracting toxic substances is treated the effect of skin ulcer.
The present invention is based in glycolytic cycle; Hexokinase catalysis ATP is converted into the reaction of ADP; ATP is mixed with Chinese medicine extract under certain condition, is means with the LC-MS method, (adds or do not add Chinese medicine extract) before and after the assaying reaction respectively; The amount of the variable quantity of ATP and generation ADP suppresses active in order to the hexokinase of estimating Chinese medicine extract.In the implementation process of LC-MS method, in mobile phase, add the 5mM ammonium acetate, the mass signal that has greatly improved ATP reaches the quantitative stability of ATP.
The selection result shows, the screening technique of the inhibiting activity extract of the auxiliary tool hexokinase of LC-MS method of the present invention is simple to operate, automaticity is high, highly sensitive and screening fast.
This method need not to carry out large batch of zoopery, compares test cell line, and extract consumption of the present invention is few, biochemical reagents save and the screening cycle short, for the screening of active component in the Chinese crude drug with study significant.The experiments parameter has good reference value for the biomolecule of utilizing mass-spectrometric technique to measure other similarity among the LC-MS that warp is optimized among the present invention.
The inventive method is fast and convenient, and economical and effective is fit to the screening of the inhibiting active component of tool hexokinase activity in the Chinese crude drug.
The inventive method can be used for the detection of ATP in the various complex matrices.
Description of drawings
Fig. 1. typical ATP chromatogram,
Wherein, on: be respectively ATP and blank, down: ADP and blank
Fig. 2. the hexokinase Michaelis constant is calculated.
Fig. 3. the Semen Momordicae extract chromatographic fractionation figure.
Fig. 4. Semen Momordicae extract suppresses hexokinase typical color spectrogram,
Among the figure, from left to right be ATP, ADP;
Each peak is represented: blank group, enzyme inhibition group, enzymatic reaction group.
The specific embodiment
Embodiment 1: hexokinase suppresses the screening of active component in the Semen Momordicae extract
1.LC-MS method is measured ATP and ADP
Chromatographic column: Agilent Extend C-18 (3.1 * 100mm, 3 μ m; Agilent, Palo Alto, CA, USA); Mobile phase: 5mM ammonium acetate (pH7.5): methanol=6: 4; Flow velocity: 0.3mL/min; Column temperature: 20 ℃; Sample size: 10 μ L
Ion source: liquid chromatograph mass spectrography interface: electrospray interface (ESI), positive ion mode (positive ion); Dry gas flow velocity: 10mL/min; Dry gas temperature: 300 ℃; Atomization gas pressure: 30psi (upper limit 60psi); Capillary voltage: 3000V; Mass analyzer: detection mode: select ion monitoring (SIM); Between detection zone: 0-1.6min; Fragment voltage: 100V; Monitoring ion: ATP, quasi-molecular ion peak (m/z 508); ADP, quasi-molecular ion peak (m/z428)
Specificity is as shown in Figure 1, and the ATP retention time is 1.39 minutes, and the ADP retention time is 1.40 minutes, can know with blank chromatogram contrast, and going out the peak position does not have obvious impurity interference.The result shows that with the quantitative ATP of this method, specificity is good.
The range of linearity with the theoretical concentration X of ATP peak area Y to ATP, is that weight coefficient is done the weighted linear recurrence with 1/x in 5-60 μ g/mL scope, gets the standard curve equation to be: Y=3022.6X+1902.9 (r=0.998).
Sensitivity this method is 5 μ g/mL (S/N >=10) to the minimum quantitative limit (LLOQ) of ATP, and LDL (LLOD) is 1 μ g/mL (LLOD).LLOQ the sky in and day between precision be respectively 3.95% and 11.36%, accuracy is respectively 105.37% and 101.09%.
Each 5 parts of basic, normal, high 3 the concentration quality-control samples of precision and accuracy preparing A TP (12,35,55 μ g/mL), the sample introduction analysis, and with standard curve calculating on the same day day an interior precision and an accuracy.METHOD FOR CONTINUOUS DETERMINATION is 3 days in accordance with the law, calculates precision and accuracy between the sky.The result shows (as shown in table 1), the basic, normal, high concentration quality-control sample of this method, and precision is all less than 10% in day and between the sky, and accuracy is within 100 ± 5%.
The accuracy and the precision of table 1:ATP quality-control sample
2. mensuration (the Michaelis constant K of hexokinase activity
mMensuration)
The preparation of ATP series solution with substrate buffer solution (precision takes by weighing HEPES 300.0mg, magnesium chloride 60.0mg, anhydrous glucose 50.0mg, the 50ml deionized water fully dissolves, with 1mol/L sodium hydroxide accent pH to 7.5, must 25mM HEPES buffer.This buffer solution fresh every day) the ATP storing solution is diluted to 5,10,15,20 respectively, 25,30,35 μ g/mL series solution supplies enzymatic reaction to use.
The hexokinase enzymatic reaction adds 100 μ L hexokinase buffer respectively in the 1.5mL centrifuge tube (precision takes by weighing HEPES 300.0mg; Magnesium chloride 60.0mg; The 50ml deionized water fully dissolves, and adds 5% (m/v) glycerol, transfers pH to 7.5 with the 1mol/L sodium hydroxide; Get 25mM HEPES buffer) and 100 μ LATP series solution, sample introduction behind the vortex 10s.In different centrifuge tubes, add 100 μ L substrate buffer solutions and 100 μ LATP series solution in addition respectively, as the blank group.
Hexokinase Michaelis constant Km calculates Michaelis-Menten equation: 1/V=K
m/ Vm * 1/ [S]+1/Vm, wherein 1/V is a response speed, i.e. ATP reduction or ADP growing amount (this experiment with ATP reduction calculate), [S] is concentration of substrate, i.e. ATP concentration, unit conversion does.As vertical coordinate, the concentration of ATP is as abscissa with the inverse of the difference of ATP peak area in ATP peak area and the enzymatic reaction group in the corresponding concentration blank group, and mapping (Fig. 2) is then schemed the cathetus slope and is hexokinase K
mCalculate K with method of least square
mBe 0.36.
3. the screening of hexokinase inhibitor activity component in the Semen Momordicae extract
The preparation 500g Semen Momordicae of the Semen Momordicae extract pulverizing that shells, in the 500ml petroleum ether, reflux is soaked after 1 hour and was removed degrease, triplicate in 24 hours.Filtered filtration residue was extracted 24 hours with the 500ml soaked in absolute ethyl alcohol, repeated to extract merge extractive liquid, three times.Low-temperature centrifugation is removed in the extracting solution behind the impurity, and the evaporate to dryness ethanol that circles round is processed fluid extract.Take by weighing Semen Momordicae extract and heat up in a steamer extractum 500mg and be dissolved in 5ml 50% ethanol, fully obtain yellow solution after the dissolving, a small amount of insoluble impurity is fallen in centrifugal filtration, is made into the 100mg/mL mother solution, separates with HPLC.The separate colors spectrogram is as shown in Figure 3.(4.00～7.00min), (16.00～20.00min), (23.00～25.50min), MBZD (27.00～28.00min) for MBZC for MBZB to collect effluent and called after MBZA respectively.Merge and collect liquid, make four kinds of extract dry powder behind the evaporate to dryness that circles round.
Solution preparation takes by weighing an amount of Semen Momordicae extract MBZA, MBZB, MBZC and MBZD dry powder and is made into the solution of 30 μ g/ml, the insoluble composition of centrifugal filtration respectively with substrate buffer solution.Compound concentration is 30 μ g/mL ATP solution for later use in addition.
Each sample introduction was analyzed for five times after each solution of table 3 preparation was pressed in the screening of hexokinase inhibition activity extract.Behind the sample introduction; With the ATP peak area ratio of the meansigma methods that adds gained ATP peak area behind the Semen Momordicae extract sample feeding five times and blank sample and enzymatic reaction control sample (unrestraint agent); As shown in table 4; The ATP peak area of the ATP peak area that adds the Semen Momordicae extract sample in the enzymatic reaction sample, explaining to contain among the MBZA has inhibiting active component to hexokinase.
Each solution preparation of table 3. extract screening
Each extract A TP peak area of table 4. Semen Momordicae
MBZA suppresses active mensuration and calculates precision to take by weighing 10.0mgMBZA in the 10ml volumetric flask, is settled to scale with substrate buffer solution, processes mother solution.Continuation is diluted to 10,15 with substrate buffer solution with the MBZA mother solution, 20,25,30,50,80,100 μ g/mL series solution for later use.Respectively organize solution by preparation shown in the table 3, sample introduction gets chromatogram respectively, MBZA blank, enzymatic reaction, enzyme inhibitory reaction group typical color spectrogram such as Fig. 4, and this moment, the concentration of MBZA was 50 μ g/mL.
Claims (6)
1. the screening technique of the inhibiting active ingredient of Chinese herbs of tool hexokinase; It is characterized in that, be the hexokinase catalytic reaction of substrate with ATP, through the quantitative analysis to ATP and product A DP; Filter out the inhibiting active component of tool hexokinase in the Chinese crude drug, it comprises step:
(1) set up high performance liquid chromatogram-mass spectrometry combination method (LC-MS),
In mobile phase, add the ammonium acetate of 5mM, ATP and product A DP are detected and quantitative analysis;
(2) based on high performance liquid chromatogram-mass spectrometry combination method ATP is carried out quantitative analysis, measure with the hexokinase activity of ATP as substrate;
The Chinese medicine extract of the tool hexokinase inhibitor activity that (3) screening is obtained is measured and is calculated.
2. by the described method of claim 1, it is characterized in that, in the said step (1) to the minimum 5 μ g/mL that quantitatively are limited to of polar molecule ATP.
3. by the described method of claim 1, it is characterized in that in the said step (1), mobile phase is added the ammonium acetate of 5mM, is detector with the mass spectrum, capillary voltage is set to 3000V, and the monitoring ion is respectively ATP:m/z 508, and ADP:m/z 428.
4. by the described method of claim 1, it is characterized in that, in the said step (1), ATP and product A DP detected and quantitative analysis:
Chromatographic column: Agilent Extend C-18 (3.1 * 100mm, 3 μ m; Agilent, Palo Alto, CA, USA); Mobile phase: 5mM ammonium acetate (pH7.5): methanol=6: 4; Flow velocity: 0.3mL/min; Column temperature: 20 ℃; Sample size: 10 μ L;
Ion source: liquid chromatograph mass spectrography interface: electrospray interface (ESI), positive ion mode (positive ion); Dry gas flow velocity: 10mL/min; Dry gas temperature: 300 ℃; Atomization gas pressure: 30psi (upper limit 60psi); Capillary voltage: 3000V; Mass analyzer: detection mode: select ion monitoring (SIM); Between detection zone: 0-1.6min; Fragment voltage: 100V; Monitoring ion: ATP, quasi-molecular ion peak (m/z 508); ADP, quasi-molecular ion peak (m/z428).
5. by the described method of claim 1, it is characterized in that in the said step (3), Chinese medicine extract is four kinds of extract MBZA, MBZB, MBZC and MBZD of Chinese crude drug Semen Momordicae.
6. by the described method of claim 1, it is characterized in that in the said step (3), the Chinese medicine extract MBZA of the tool hexokinase inhibitor activity that screening is obtained measures and calculates.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102796805A (en) * | 2012-08-10 | 2012-11-28 | 中国科学院长春应用化学研究所 | Method for screening cyclin-dependent kinases 5 inhibitor |
| CN110967451A (en) * | 2019-12-23 | 2020-04-07 | 农业农村部环境保护科研监测所 | Method for rapidly identifying cadmium accumulation of rice by utilizing in-vitro rice ears |
| CN114948920A (en) * | 2021-04-21 | 2022-08-30 | 苏州大学 | Application of small molecular compound in preparation of antitumor drugs |
-
2010
- 2010-07-26 CN CN2010102385104A patent/CN102335221A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| 《Chromatographia》 20100701 Le Pan et al An LC-MS Method for a Hexokinase Inhibitor Study Based on Adenosine 5'-Triphosphate Determination and Application to the Anticancer Mechanism of Momordica cochinchinensis 第425-430页 1-3,6 , * |
| LE PAN ET AL: "An LC–MS Method for a Hexokinase Inhibitor Study Based on Adenosine 5’-Triphosphate Determination and Application to the Anticancer Mechanism of Momordica cochinchinensis", 《CHROMATOGRAPHIA》 * |
| 张丹等: "木鳖子提取液抗氧化活性的分析 ", 《复旦学报(医学版)》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102796805A (en) * | 2012-08-10 | 2012-11-28 | 中国科学院长春应用化学研究所 | Method for screening cyclin-dependent kinases 5 inhibitor |
| CN102796805B (en) * | 2012-08-10 | 2013-11-27 | 中国科学院长春应用化学研究所 | Method for screening cyclin-dependent kinases 5 inhibitor |
| CN110967451A (en) * | 2019-12-23 | 2020-04-07 | 农业农村部环境保护科研监测所 | Method for rapidly identifying cadmium accumulation of rice by utilizing in-vitro rice ears |
| CN114948920A (en) * | 2021-04-21 | 2022-08-30 | 苏州大学 | Application of small molecular compound in preparation of antitumor drugs |
| WO2022222261A1 (en) * | 2021-04-21 | 2022-10-27 | 苏州大学 | Hexokinase 2 inhibitor screening method and application of small molecule compound in preparation of antitumor drug |
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Application publication date: 20120201 |