A method for rapid screening of compounds with anti-tumor metastatic activity

Abstract

The application provides a method for rapidly screening anti-tumor metastasis active compounds, relates to the technical field of tumor treatment, and uses a statistical data analysis method to rapidly screen active monomer compounds for resisting tumor metastasis from traditional Chinese medicines containing several hundred compounds, and based on the method, nine components are extracted from traditional Chinese medicine forsythia suspensa; the scratch activity group of the nine components is combined with compound information obtained through mass spectrum analysis; and through statistical data analysis, the active monomer compound verbascoside VB contained in forsythia suspensa for inhibiting tumor metastasis is rapidly screened.

Description

Technical Field

[0001] This invention relates to the field of tumor treatment technology, and to a method for rapidly screening compounds with anti-tumor metastasis activity. Background Technology

[0002] Tumor metastasis is one of the main causes of treatment failure and poor prognosis in cancer patients, making the development of drugs targeting tumor metastasis crucial. Traditional Chinese medicine (TCM), a treasure trove of natural products, contains compounds with novel structures and diverse activities, making it an important source for anti-tumor drug development. However, TCM typically contains hundreds of compounds, and traditional methods for discovering active ingredients rely on repeated extraction, separation, purification, and structural identification, followed by individual activity testing. This process is cumbersome, time-consuming, and inefficient, and some compounds with low content but good activity are difficult to obtain using traditional methods. Therefore, there is an urgent need to establish a method for rapidly and efficiently screening anti-tumor metastasis active compounds from the complex TCM system.

[0003] By analyzing the chemical composition of complex mixtures using UPLC-MS / MS systems, and combining this with multivariate data analysis (such as orthogonal partial least squares discriminant analysis-OPLS-DA), chemical composition profiles can be correlated with bioactivity results without individual separation, rapidly identifying candidate compounds that contribute the most to activity. However, the application of this method in the screening of active ingredients for antitumor metastasis is currently lacking. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for rapidly screening active compounds against tumor metastasis, which can rapidly screen active monomeric compounds against tumor metastasis from traditional Chinese medicine containing hundreds of compounds.

[0005] The technical solution adopted by this invention to achieve its technical objectives is as follows:

[0006] A method for rapidly screening compounds with anti-tumor metastasis activity includes:

[0007] 1) The traditional Chinese medicine was extracted in multiple stages according to its polarity from low to high. All extracted components were analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) and tumor cell scratch activity test to obtain the compound mass spectrometry information and anti-tumor migration activity of each component.

[0008] 2) Calculate the anti-tumor migration score of each component, and take the components with scores above the median as the active group and the remaining components as the inactive group;

[0009] 3) Combine the activity grouping with the mass spectrometry information of each component, and obtain the contribution of each compound to the activity group, i.e., the VIP value, through OPLS-DA analysis. Sort the VIP values ​​and screen out a series of compounds with large VIP values, i.e., candidate anti-tumor metastasis active compounds.

[0010] 4) Match the mass-to-charge ratio (m / z) of the selected candidate compounds to the database of traditional Chinese medicines to obtain the name, molecular formula, molecular weight, and chemical structure of the candidate compounds. Under the same injection conditions as the previous component mass spectrometry analysis, perform UPLC-MS / MS analysis on the standard solutions of each candidate compound to obtain the mass spectrometry information of the candidate compounds. Compare the mass spectrometry information of the components to confirm the candidate compounds. At the same time, perform tumor cell scratch assay on the standard solutions of the candidate compounds to obtain the anti-migration activity of the candidate compounds. Confirm the candidate compounds based on activity. Combining mass spectrometry and activity analysis, the candidate compounds are finally confirmed.

[0011] Preferably, in step 1), the scratch area is obtained through a tumor cell scratch activity test, and the migration inhibition rate of each component on tumor cells is further calculated:

[0012] x-hour migration inhibition rate = (1 - x-hour migration rate of drug-treated group / x-hour migration rate of blank treatment group) 100%;

[0013] Migration rate at x hours = (1 - scratch area at x hours / scratch area at 0 hours) 100%.

[0014] Preferably, in step 2), the anti-tumor migration score of each component is calculated using a migration inhibition rate scoring system:

[0015] Scoring formula = , The migration inhibition rate is given at a certain concentration over a certain period of time. (Three concentrations) , , All components were at the IC50 level for their anti-proliferation effects on tumor cells. 50 At concentrations below [specific concentration].

[0016] Preferably, in step 3), an S-plot is obtained through OPLS-DA analysis, and discrete points on one side are circled. A Biplot is obtained through activity analysis, showing that the circled discrete points are on the side of the active group. Therefore, the circled discrete points are active monomeric compounds that contribute more to the active group, that is, monomeric compounds with large VIP values. The group contribution values ​​of the circled discrete points are sorted in descending order of VIP values.

[0017] Preferably, the traditional Chinese medicine is Forsythia suspensa; Forsythia suspensa powder is extracted with ultrapure water to obtain Forsythia suspensa aqueous extract LQ1, and then extracted with petroleum ether, ethyl acetate and n-butanol sequentially to obtain petroleum ether extract fraction LQ2, ethyl acetate extract fraction LQ3, n-butanol extract fraction LQ4 and residual fraction LQ5 after extraction of Forsythia suspensa aqueous extract; Forsythia suspensa powder is extracted with ethanol to obtain Forsythia suspensa ethanol extract LQ6, and then extracted with petroleum ether and ethyl acetate sequentially to obtain petroleum ether extract fraction LQ7, ethyl acetate extract fraction LQ8 and residual fraction LQ9 after extraction of Forsythia suspensa ethanol extract.

[0018] More preferably, forsythia powder is added to ultrapure water at a material-to-liquid ratio of 1:10 (g / mL) and boiled at 80°C for one hour. After filtering the residue, forsythia water extract is obtained. Forsythia powder is added to 90% ethanol at a material-to-liquid ratio of 1:10 (g / mL) and refluxed at 60°C for half an hour with stirring. After filtering the residue, forsythia ethanol extract is obtained.

[0019] More preferably, MDA-MB-231 breast cancer cells with a normal growth rate, at 1.8 10 4 Cells were seeded into 96-well plates and incubated in an incubator. Once the cell density reached 90%, a 96-well scratcher was used to scratch the cells. The culture medium in the wells was discarded, and the plates were washed once with PBS. The PBS was discarded, and then DMEM basal medium containing 0.5% serum was added. The plates were then placed in a microplate imaging system to obtain images after 0 hours of drug treatment. Nine components of Forsythia were prepared into solutions with a concentration of 20 mg / mL and diluted 5 / 6 times to obtain nine different concentration groups and a blank control group without any component solution. Each component had three replicates, and 10 μL of solution was added to each concentration well, resulting in drug treatment concentrations of 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.031, 0.016, 0.008, and 0. mg / mL, and then placed in an incubator for incubation. Scratch images were taken at 12, 24, 36, and 48 hours after drug treatment. The scratch area was obtained after processing, and the migration inhibition rate at each concentration for 12, 24, 36, and 48 hours was obtained.

[0020] More preferably, t(xh) represents time (12h, 24, 36h, 48h) and C(i) represents concentration (C1 8μg / mL, C2 16μg / mL, C3 32μg / mL).

[0021] More preferably, the antitumor metastasis active compound finally screened from Forsythia suspensa is verbascoside VB.

[0022] The beneficial effects of this invention are as follows:

[0023] This invention proposes a method for rapidly screening compounds with anti-tumor metastasis activity. Using statistical data analysis, active monomeric compounds with anti-tumor metastasis activity are rapidly screened from traditional Chinese medicine containing hundreds of compounds. Furthermore, based on this method, nine components were extracted from the traditional Chinese medicine Forsythia suspensa. The scratch activity grouping of these nine components was combined with compound information obtained from mass spectrometry analysis. Through statistical data analysis, verbascoside VB, an active monomeric compound in Forsythia suspensa that inhibits tumor metastasis, was rapidly screened. Attached Figure Description

[0024] Figure 1 Total ion chromatogram of Forsythia suspensa AE fraction analyzed by UPLC-MS / MS.

[0025] Figure 2 Total ion chromatogram of PEAE fraction of Forsythia suspensa analyzed by UPLC-MS / MS.

[0026] Figure 3 Total ion chromatogram of Forsythia suspensa EAAE fraction analyzed by UPLC-MS / MS.

[0027] Figure 4 Total ion chromatogram of NBAE component of Forsythia suspensa analyzed by UPLC-MS / MS.

[0028] Figure 5 Total ion chromatogram of WAE components of Forsythia suspensa analyzed by UPLC-MS / MS.

[0029] Figure 6 Total ion chromatogram of Forsythia suspensa EE fraction analyzed by UPLC-MS / MS.

[0030] Figure 7 Total ion chromatogram of PEEE components of Forsythia suspensa analyzed by UPLC-MS / MS.

[0031] Figure 8 Total ion chromatogram of Forsythia suspensa EAEE fraction analyzed by UPLC-MS / MS.

[0032] Figure 9 Total ion chromatogram of EtEE component of Forsythia suspensa analyzed by UPLC-MS / MS.

[0033] Figure 10 Migration inhibition rate of extracts from various components of Forsythia suspensa.

[0034] Figure 11 Migration inhibition rate scores of extracts from different components of Forsythia suspensa.

[0035] Figure 12 Score chart of OPLS-DA analysis of extracts from various components of Forsythia suspensa.

[0036] Figure 13 S-plot of OPLS-DA analysis of extracts from various components of Forsythia suspensa.

[0037] Figure 14 Biplot of OPLS-DA analysis of extracts from various components of Forsythia suspensa.

[0038] Figure 15 VIP values ​​of OPLS-DA analysis of extracts from various components of Forsythia suspensa.

[0039] Figure 16 The cleavage mechanism of verbascoside.

[0040] Figure 17 Structural diagram of verbascoside, chromatogram of 4449 and primary and secondary mass spectra.

[0041] Figure 18 Spectrum of standard quality of verbascoside.

[0042] Figure 19 Secondary mass spectrum of verbascoside standard.

[0043] Figure 20 Verbascoside cell activity.

[0044] Figure 21 Verbascoside inhibits cell scratch healing.

[0045] Figure 22 Migration inhibition rate of verbascoside in scratch assay.

[0046] Figure 23 Migration inhibition rates of verbascoside and compounds contained in Forsythia suspensa at 30 μg / mL; QR (Quercetin), CA (Chlorogenic acid), PL (Pinoresinol), Hy (Hyperoside), AG (Arctigenin), RT (Rutin), PM ((+) pinoresinol monomethylether-β-D-glucoside, (+) pinoresinol monomethylether-4-O-β-D-glucoside), AR (Arctiin), PG (Phillygenin). Detailed Implementation

[0047] The present invention will now be described in detail with reference to specific embodiments. The following specific embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way.

[0048] Example

[0049] Taking the traditional Chinese medicine Forsythia suspensa as an example, this invention provides a method for rapidly screening compounds with anti-tumor metastasis activity, specifically including:

[0050] (1) Search literature on Forsythia compounds and establish a Forsythia compound library, including compound name, molecular formula, precise molecular weight and precise mass-to-charge ratio.

[0051] (2) Forsythia powder was added to ultrapure water at a material-to-liquid ratio of 1:10 (g / mL) and boiled at 80℃ for one hour (stirring the residue once every ten minutes). After filtering the residue, a water extract of Forsythia was obtained (LQ1, AE). The water extract of Forsythia was extracted sequentially with petroleum ether, ethyl acetate, and n-butanol to obtain the petroleum ether extract fraction (LQ2, PEAE), ethyl acetate extract fraction (LQ3, EAAE), n-butanol extract fraction (LQ4, NBAE), and the residual fraction after extraction of the water extract of Forsythia (LQ5, WAE). Forsythia powder was added to 90% ethanol at a material-to-liquid ratio of 1:10 (g / mL) and refluxed at 60℃ for half an hour with stirring. After filtering the residue, an ethanol extract of Forsythia was obtained (LQ6, EE). The ethanol extract of Forsythia was extracted sequentially with petroleum ether and ethyl acetate to obtain the petroleum ether extract fraction (LQ7, PEEE) and ethyl acetate extract fraction (LQ8, EAEE). And the residual components after extraction of the ethanol extract of Forsythia suspensa (LQ9, EtEE); a total of 9 components were obtained from the extraction and extraction of Forsythia suspensa powder.

[0052] (3) The nine components in step (2) were detected by ultra-high performance liquid chromatography-quadrupole electrostatic field orbital trap mass spectrometry (UPLC-Q-ExactiveOrbitrap-MS / MS) to obtain the mass spectrometric information of the compounds contained in each of the nine components. Figures 1-9 The total ion current chromatogram of each component extract is shown.

[0053] (4) The nine components from step (2) were subjected to tumor cell scratch assays. MDA-MB-231 breast cancer cells with normal growth rate were subjected to a scratch assay at 1.8... 10 4Cells were seeded into 96-well plates and incubated. After approximately 24 hours of incubation, when the cell density reached 90%, a 96-well scratcher was used to scratch the cells. The culture medium in the wells was discarded, and the plates were washed once with PBS. The PBS was discarded, and then 90 μL of DMEM basal medium containing 0.5% serum was added. The plates were then placed in a Cytation 1 instrument to obtain images at 0 hours of drug treatment. Nine components of Forsythia were prepared into solutions with a concentration of 20 mg / mL and diluted 1 / 2 times to obtain nine different concentration groups and a blank control group without any component solution. Each component had three replicates, with 10 μL of solution added to each well, resulting in drug treatment concentrations of 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.031, 0.016, 0.008, and 0 mg / mL. The plates were then incubated, and scratch images were taken at 12, 24, 36, and 48 hours of drug treatment. ImageJ processes all scratched photos to obtain the scratch area. The migration rate at x hours is calculated as (1 - scratch area at x hours / scratch area at 0 hours). 100%, for example, 12-hour migration rate = (1 - 12-hour area / 0-hour area) 100%, and using this formula, the migration rates at each concentration for 12, 24, 36, and 48 hours were obtained. x-hour migration inhibition rate = (1 - x-hour migration rate of the drug-treated group / x-hour migration rate of the blank treatment group) 100%, for example, 12-hour migration inhibition rate = (1-12-hour migration rate of the drug-treated group / x-hour migration rate of the blank-treated group) 100%, and using this formula, the migration inhibition rates at each concentration for 12, 24, 36, and 48 hours were obtained. Therefore, the migration inhibition rate of each component on MDA-MB-231 breast cancer cells was obtained ( Figure 10 ).

[0054] (5) Using the migration inhibition rate scoring system, the nine components in step (2) are scored according to the migration inhibition rate obtained in step (4). The scores of each component are sorted from high to low. The components with a median score or higher are classified as the Active group, and the remaining components are classified as the Inactive group. Figure 11 ).

[0055] Scoring formula = , The migration inhibition rate at a certain concentration over a certain time period. For times of 12h, 24, 36h, and 48h, The concentrations were C1 8 μg / mL, C2 16 μg / mL, and C3 32 μg / mL. These three concentrations were all within the IC50 range for each component's anti-proliferation activity against tumor cells. 50At concentrations below a certain level, the inhibitory activity of each component on tumor cell migration was determined by eliminating the influence of each component on tumor cell proliferation inhibition, resulting in a more accurate anti-migration activity.

[0056] (6) Combine the compound information of each of the nine components in step (3) with the grouping information in step (5), and analyze them using OPLS-DA. Figure 12 ) to obtain the S-plot, circle the discrete points on one side ( Figure 13 The activity analysis yielded a Biplot (). Figure 14 The selected discrete points are located on the side of the active group, therefore, the selected discrete points are the active monomer compounds that contribute significantly to the active group, i.e., monomer compounds with high VIP values. The group contribution values ​​of the selected discrete points are then sorted in descending order of VIP value. Figure 15 The table lists the retention times and mass-to-charge ratios of the active monomer compounds (Table 1).

[0057] Table 1. Top 5 Potential Antitumor Active Ions by VIP Value

[0058]

[0059] (7) The mass-to-charge ratio of the candidate compound with the highest VIP value in step (6) is searched and matched in the Forsythia compound library in step (1) to obtain candidate compound information (Table 2).

[0060] Table 2 Identification of compounds with antitumor invasion and metastasis activity

[0061]

[0062] The standard was analyzed by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) and scratch assay. By comparing retention time and secondary mass spectrometry fragments, the selected compound that inhibited migration was identified as verbascoside VB. Figure 16-19 Verbascoside VB was subjected to cell viability assays (MTT) and scratch assays (…). Figure 20-22 The migration inhibition rate of 30 μg / mL verbascoside VB was compared with that of nine common compounds in Forsythia suspensa. The results showed that the migration inhibition activity of verbascoside VB was 41.47%, which was higher than that of the nine compounds. Figure 23 ).

[0063] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for rapidly screening compounds with anti-tumor metastasis activity, comprising: 1) The traditional Chinese medicine was extracted in multiple stages according to its polarity from low to high. All extracted components were analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry and tumor cell scratch activity test to obtain the compound mass spectrometry information and anti-tumor migration activity of each component. 2) Calculate the anti-tumor migration score of each component, and take the components with scores above the median as the active group and the remaining components as the inactive group; 3) Combine the activity grouping with the mass spectrometry information of each component, and obtain the contribution of each compound to the activity group, i.e., the VIP value, through OPLS-DA analysis. Sort the VIP values ​​and screen out a series of compounds with large VIP values, i.e., candidate anti-tumor metastasis active compounds. 4) Match the mass-to-charge ratio (m / z) of the selected candidate compounds to the database of traditional Chinese medicines to obtain the name, molecular formula, molecular weight, and chemical structure of the candidate compounds. Under the same injection conditions as the previous component mass spectrometry analysis, perform UPLC-MS / MS analysis on the standard solutions of each candidate compound to obtain the mass spectrometry information of the candidate compounds. Compare the mass spectrometry information of the components to confirm the candidate compounds. At the same time, perform tumor cell scratch assay on the standard solutions of the candidate compounds to obtain the anti-migration activity of the candidate compounds. Confirm the candidate compounds based on activity. Combining mass spectrometry and activity analysis, the candidate compounds are finally confirmed.

2. The method for rapidly screening compounds with anti-tumor metastasis activity according to claim 1, characterized in that, In step 1), the scratch area is obtained through a tumor cell scratch activity test, and the migration inhibition rate of each component on tumor cells is further calculated: x-hour migration inhibition rate = (1 - x-hour migration rate of drug-treated group / x-hour migration rate of blank treatment group) 100%; Migration rate at x hours = (1 - scratch area at x hours / scratch area at 0 hours) 100%.

3. The method for rapidly screening compounds with anti-tumor metastasis activity according to claim 2, characterized in that, In step 2), the anti-tumor migration score of each component is calculated using a migration inhibition rate scoring system: Scoring formula = , The migration inhibition rate is given at a certain concentration over a certain period of time. (Three concentrations) , , All components were at the IC50 level for their anti-proliferation effects on tumor cells. 50 At concentrations below [specific concentration].

4. A method for rapidly screening compounds with anti-tumor metastasis activity according to claim 1 or 3, characterized in that, In step 3), an S-plot is obtained through OPLS-DA analysis. Discrete points on one side are circled. A Biplot is obtained through activity analysis, showing that the circled discrete points are on the side of the active group. Therefore, the circled discrete points are active monomeric compounds that contribute more to the active group, that is, monomeric compounds with large VIP values. The group contribution values ​​of the circled discrete points are sorted in descending order of VIP value.

5. The method for rapidly screening compounds with anti-tumor metastasis activity according to claim 3, characterized in that, The traditional Chinese medicine is Forsythia suspensa. Forsythia suspensa powder was extracted with ultrapure water to obtain Forsythia suspensa aqueous extract LQ1. The Forsythia suspensa aqueous extract was then extracted sequentially with petroleum ether, ethyl acetate, and n-butanol to obtain petroleum ether extract fraction LQ2, ethyl acetate extract fraction LQ3, n-butanol extract fraction LQ4, and residual fraction LQ5 after extraction of the Forsythia suspensa aqueous extract. Forsythia suspensa powder was extracted with ethanol to obtain Forsythia suspensa ethanol extract LQ6. The Forsythia suspensa ethanol extract was then extracted sequentially with petroleum ether and ethyl acetate to obtain petroleum ether extract fraction LQ7, ethyl acetate extract fraction LQ8, and residual fraction LQ9 after extraction of the Forsythia suspensa ethanol extract.

6. The method for rapidly screening compounds with anti-tumor metastasis activity according to claim 5, characterized in that, Forsythia powder was added to ultrapure water at a ratio of 1:10 g / mL and boiled at 80°C for one hour. After filtering the residue, a water extract of Forsythia was obtained. Forsythia powder was added to 90% ethanol at a ratio of 1:10 g / mL and refluxed at 60°C for half an hour with stirring. After filtering the residue, an ethanol extract of Forsythia was obtained.

7. The method for rapidly screening compounds with anti-tumor metastasis activity according to claim 5, characterized in that, MDA-MB-231 breast cancer cells with a normal growth rate, at 1.8 10 4 Cells were seeded into 96-well plates and incubated in an incubator. Once the cell density reached 90%, a 96-well scratcher was used to scratch the cells. The culture medium in the wells was discarded, and the plates were washed once with PBS. The PBS was discarded, and then DMEM basal medium containing 0.5% serum was added. The plates were then placed in a microplate imaging system to obtain images after 0 hours of drug treatment. Nine components of Forsythia were prepared into solutions with a concentration of 20 mg / mL and diluted 5 / 6 times to obtain nine different concentration groups and a blank control group without any component solution. Each component had three replicates, and 10 μL of solution was added to each concentration well, resulting in drug treatment concentrations of 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.031, 0.016, 0.008, and 0. mg / mL, and then placed in an incubator for incubation. Scratch images were taken at 12, 24, 36, and 48 hours after drug treatment. The scratch area was obtained after processing, and the migration inhibition rate at each concentration for 12, 24, 36, and 48 hours was obtained.

8. The method for rapidly screening compounds with anti-tumor metastasis activity according to claim 5, characterized in that, t(xh) represents time (12h, 24h, 36h, 48h), and C(i) represents concentration (C1 8μg / mL, C2 16μg / mL, C3 32μg / mL).

9. The method for rapidly screening compounds with anti-tumor metastasis activity according to claim 7, characterized in that, The anti-tumor metastasis active compound finally screened from Forsythia suspensa was verbascoside VB.

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