Method for screening anti-colon cancer active ingredients of traditional Chinese medicine based on exosome affinity chromatography technology

Abstract

The invention provides a method for screening traditional Chinese medicine anti-colon cancer active ingredients by adopting an exosome affinity chromatography technology, and belongs to the technical field of medicine screening. The method comprises the following steps: 1, adding an aldehyde group on amino silicon dioxide by using glutaraldehyde, co-incubating with an exosome derived from cancer cells, and closing the redundant aldehyde group by using lysine to form an exosome affinity chromatography carrier material; 2, co-incubating a traditional Chinese medicine extract to be screened and the affinity chromatography carrier material for a certain time, centrifugally absorbing an unadsorbed solution, and eluting with a proper solvent to obtain an eluent; the method comprises the following steps: comparing peak area changes of a traditional Chinese medicine extract initial solution, a non-adsorbed solution and an eluent by adopting high performance liquid chromatography, so as to screen out traditional Chinese medicine components having an adsorption effect with exosomes; 3, identifying the screened traditional Chinese medicine components by adopting a liquid chromatography-mass spectrometry technology; and 4, determining the inhibition effect of the traditional Chinese medicine components on the growth of colon cancer cells by adopting a cell experiment, thereby efficiently and accurately screening the anti-colon cancer active components.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug screening, and in particular relates to a method for screening anti-colon cancer active ingredients of traditional Chinese medicine based on exosome affinity chromatography technology. Background Art

[0002] Exosomes, as natural carriers of intercellular information exchange, play an important role in a variety of physiological processes and diseases, such as protein quality control, development, immunity, tissue homeostasis, cancer and neurodegenerative diseases. At the same time, due to their excellent biocompatibility, low immunogenicity, efficient drug loading and targeted delivery capabilities, exosomes have attracted much attention in the treatment of inflammatory bowel disease. Tumor-derived exosomes (TDEs) have been regarded as excellent natural drug carriers in the field of tumor treatment due to their unique "homing" ability. Through the specific proteins and nucleic acids they carry, TDEs can regulate immune cell activity and transmit inhibitory signals, thereby significantly affecting the response of the immune system. In addition, viruses also use the exosome biogenesis pathway to assemble infectious particles and establish host susceptibility. Based on these unique characteristics, exosomes are being developed as therapeutic agents in a variety of disease models.

[0003] Affinity chromatography is a liquid chromatography method that uses a biologically relevant binding agent as a stationary phase to separate and purify specific targets by utilizing the selective and reversible binding that occurs in many biological interactions, including the interaction between antibodies and antigens, enzymes and substrates, hormones and receptors. The selectivity and simplicity of affinity chromatography make it highly efficient for the purification of many biomolecules, biopharmaceuticals, and some other reagents. Affinity chromatography can be used not only for sample preparation, but also for the separation or measurement of biologically active components.

[0004] Colorectal cancer (CRC) is a common digestive tract malignancy that occurs in the colon. It usually originates from colon polyps and may cause digestive system dysfunction. Colorectal cancer is a major health problem worldwide and the second leading cause of cancer-related death. Traditional cancer treatments, such as surgery, radiotherapy, and chemotherapy, face limitations and side effects, and treatment options are still very limited. Therefore, there is an urgent need to develop or discover new cancer treatment drugs.

[0005] Most drugs have poor tumor targeting and lack specificity, which not only limits the therapeutic effect of the drugs, but also easily causes more serious adverse reactions. In addition, there are usually multiple immunosuppressive signals in colon cancer tumor tissues. By utilizing the characteristics of exosomes that can regulate immune cell activity and transmit inhibitory signals, the selected therapeutic drugs can improve the targeting of drugs.

[0006] Therefore, the present invention aims to construct an innovative drug screening method using affinity chromatography using exosomes as carrier materials, which is used to efficiently screen active ingredients in traditional Chinese medicine that have inhibitory effects on colon cancer cells. This study not only helps to gain a deeper understanding of the mechanism of action of exosomes, but also provides new ideas and methods for screening active ingredients that inhibit colon cancer, thereby contributing to protecting public health. Summary of the invention

[0007] In view of this, the present invention provides a method for screening anti-colon cancer active ingredients of traditional Chinese medicine based on exosome affinity chromatography technology, which can efficiently and accurately screen active ingredients that inhibit colon cancer cells.

[0008] The technical solution adopted by the present invention to solve the technical problem is as follows: 1. A method for screening anti-colon cancer active ingredients of traditional Chinese medicine based on exosome affinity chromatography technology, comprising the following steps: (1) Preparation of exosome affinity chromatography carrier material: First, glutaraldehyde and aminosilica dispersed in PBS buffer were shaken in an oscillator at room temperature for a certain period of time to add aldehyde groups to the aminosilica. After centrifugation, the mixture was washed and filtered, and then colon cancer cell exosomes were added and incubated for a certain period of time to make the aldehyde groups of aminosilica covalently bind to the carboxyl groups of the exosomes. Lysine was then added and shaken for a certain period of time to block the excess aldehyde groups on the silica, thereby obtaining exosome silica (TDEs@SiO2) affinity chromatography carrier material. (2) Screening of anti-colon cancer components of traditional Chinese medicine: Screening of anti-colon cancer active components of traditional Chinese medicine: Affinity chromatography was used to separate traditional Chinese medicine components that can bind to cancer cell exosomes. First, the traditional Chinese medicine extract was dissolved and added to the TDEs@SiO2 affinity chromatography carrier material obtained by (1) according to a certain concentration volume. The traditional Chinese medicine extract was incubated with the TDEs@SiO2 affinity chromatography carrier material for a certain period of time, and the unadsorbed supernatant was removed by centrifugation, which was the unadsorbed liquid. The solution bound to TDEs@SiO2 is obtained by eluting with a suitable solvent for a certain period of time, which is the eluent. The initial solution of the Chinese medicine extract, the unadsorbed solution, and the eluent are respectively injected into a high-performance liquid chromatograph for detection. By analyzing the changes in the peak areas of the components in different solutions, the components bound to the Chinese medicine and TDEs@SiO2 are screened out; the components bound to the exosomes are identified by liquid chromatography-mass spectrometry; the half-maximal inhibitory concentration of the screened and identified binding components on colon cancer cells is determined, and finally the active components with anti-colon cancer effects are obtained.

[0009] Preferably, the method for preparing the exosome affinity chromatography carrier material in step (1) is characterized in that the concentration of the Chinese herbal medicine extract solution is 0.5 mg / mL.

[0010] Preferably, the method for preparing the exosome affinity chromatography carrier material in step (1) is characterized in that the co-incubation time of the Chinese herbal medicine extract and TDEs@SiO2 is 30 minutes.

[0011] Preferably, the method for preparing the exosome affinity chromatography carrier material in step (1) is characterized in that the exosomes are derived from HT-29 colon cancer cells.

[0012] Preferably, in step (1), the preparation steps of the TDEs@SiO2 exosome affinity chromatography carrier material are as follows: aminosilica is dispersed in PBS buffer, glutaraldehyde is added and shaken for activation for 2 hours, washed with water 3 times, and then exosomes are added, and the mixture is placed in an oscillator and shaken for 4 hours to fix the exosomes on the SiO2 surface, and lysine dissolved in PBS buffer is added, and the mixture is shaken for 2 hours. The composite mixture is separated by centrifugation, and washed with water 3 times to obtain the TDEs@SiO2 carrier material, and finally dispersed and stored in PBS buffer (pH=7.4).

[0013] Preferably, the method for screening the anti-colon cancer components of traditional Chinese medicine described in step (2) is characterized in that: the solution combined with TDEs@SiO2 is obtained by eluting with a suitable solvent for 30 minutes.

[0014] Preferably, the method for screening the anti-colon cancer components of traditional Chinese medicine described in step (2) is characterized in that: the affinity chromatography method is specifically: the chromatographic column is YMC-Pack ODS-A C18 (4.6 × 250 mm), the detection wavelength is 254 nm, the mobile phase A is 0.1% phosphoric acid, the mobile phase B is acetonitrile, the gradient elution program is 0 min-95% A; 10 min-75% A; 15min-65% A; 30 min-55% A; 50 min-55% A; 60 min-95% A, the column temperature is 35 ° C, the wavelength is 220 nm, and the flow rate is 1 mL / min.

[0015] Preferably, the method for screening the anti-colon cancer components of traditional Chinese medicine described in step (2) is characterized in that: the LC-MS method is specifically as follows: the chromatographic column is C18 (3.5 μm; 2.1 mm × 50 mm), the mobile phase A is 0.1% formic acid, the mobile phase B is methanol, and the gradient elution program is 0 min-70% A; 4 min-70% A; 14 min-52% A; 24 min-52% A; 25 min-70% A; 30 min-70% A. The nebulizer pressure is 35 psi, the drying gas flow rate is 8 L / min, the gas temperature is 320°C, the sheath gas temperature is 350°C, the gas flow rate is 11 L / min, and the mass range is selected as 50-3000 (m / z).

[0016] It can be seen from the above technical scheme that the present invention provides a method for screening the anti-colon cancer active ingredients of traditional Chinese medicine based on exosome affinity chromatography technology, which belongs to the field of drug screening technology. The first step of the method is to use glutaraldehyde to add aldehyde groups to aminosilica, and then co-incubate with exosomes derived from colon cancer cells, and then block the excess aldehyde groups in aminosilica with lysine to form TDEs@SiO2 affinity chromatography carrier material; the second step is to co-incubate the Chinese medicine extract to be screened with the affinity chromatography carrier material for a certain period of time, centrifuge to remove the unadsorbed liquid, and then use a suitable solvent to elute to obtain the eluate; use high performance liquid phase analysis to compare the changes in the peak areas of the initial liquid, unadsorbed liquid, and eluate of the Chinese medicine extract, so as to screen out the Chinese medicine components that have adsorption effects on exosomes; the third step is to identify the screened Chinese medicine components by liquid chromatography-mass spectrometry technology; the fourth step is to use cell experiments to determine the inhibitory effect of the Chinese medicine components on the growth of colon cancer cells, so as to efficiently and accurately screen out the anti-colon cancer active ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on the provided drawings without creative work.

[0018] Figure 1 Schematic diagram of the method for screening anti-colon cancer active ingredients of traditional Chinese medicine based on exosome affinity chromatography technology of the present invention.

[0019] Figure 2 Bioaffinity chromatogram for screening anti-colon cancer active components in Sophora flavescens using TDEs@SiO2 affinity chromatography carrier material.

[0020] Figure 3This is the result of liquid chromatography-mass spectrometry identification of anti-colon cancer components in Sophora flavescens using TDEs@SiO2 affinity chromatography carrier material.

[0021] Figure 4 To verify the inhibitory effect of a screened ingredient on HT-29 colon cancer cells and the 48-hour half-inhibitory concentration IC 50 Determination of value. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The embodiment of the present invention discloses a method for screening anti-colon cancer active ingredients of traditional Chinese medicine based on exosome affinity chromatography technology, and records the inhibitory effect of matrine screened from Sophora flavescens on HT-29 colon cancer cells. Example 1

[0024] Preparation experiment of affinity chromatography carrier material TDEs@SiO2. Disperse 50 mg of aminosilica in 4 ml PBS buffer, add glutaraldehyde and shake for 2 hours, wash with water 3 times, then add exosomes extracted from HT-29 colon cancer, shake the mixture in an oscillator for 4 hours to fix the exosomes on the SiO2 surface, dissolve 1 g of lysine in 10 ml PBS buffer and add to the above product, shake the mixture for 2 hours, separate the composite mixture by centrifugation, wash with water 3 times to obtain TDEs@SiO2 carrier material, and finally divide it into 8 portions with 5 ml PBS buffer (pH=7.4) for storage. Example 2

[0025] Affinity chromatography was used to screen components in Sophora flavescens that can bind to colon cancer exosomes. Sophora flavescens Aiton) extract 200 μL as the initial solution. Take a portion of TDEs@SiO2 and centrifuge to remove the PBS buffer, add 200 μL of Sophora flavescens extract solution, mix and place in an oscillator for incubation for 30 minutes, centrifuge and take out the supernatant as the non-adsorbed liquid. Then add 200 μL of methanol to the remaining solid, mix well and place in an oscillator for 30 minutes, centrifuge and take out the supernatant as the eluent. The Sophora flavescens extract initial solution, non-adsorbed solution and eluent were analyzed by high performance liquid chromatography. The results are shown in Figure 2The results showed that in the HPLC of the Sophora flavescens eluate, multiple chromatographic peaks can be clearly seen, that is, the TDEs@SiO2 affinity chromatography carrier material successfully adsorbed multiple components in Sophora flavescens that can bind to exosomes. Example 3

[0026] Liquid chromatography-mass spectrometry was used to identify components in Sophora flavescens that can bind to exosomes. 200 μL of Sophora flavescens extract was added to the EP tube containing TDEs@SiO2, mixed and placed in a room temperature oscillator for 30 minutes, centrifuged to obtain the supernatant (unadsorbed liquid), and then 200 μL of methanol was added to the remaining solid, mixed and placed in a oscillator for 30 minutes, centrifuged to obtain the supernatant (eluate), and the Sophora flavescens eluate was analyzed by liquid chromatography-mass spectrometry. The results are shown in Figure 3 C, is the mass spectrum of matrine, and matrine was identified ( Figure 3 B), it was preliminarily determined that one of the components in Sophora flavescens that can bind to colon cancer exosomes is KURARINONE (English name: KURARINONE). Example 4

[0027] Liquid chromatography combined with reference substance verification further confirmed the experiment of components in Sophora flavescens that can bind to colon cancer exosomes. Add 200 μL of Sophora flavescens extract to the EP tube containing TDEs@SiO2, mix well and place in a room temperature oscillator for 30 minutes, centrifuge to obtain the supernatant (unadsorbed liquid), then add 200 μL of methanol to the remaining solid, mix well and place in an oscillator for 30 minutes, centrifuge to obtain the supernatant (eluent). In addition, prepare a sophora flavescens reference substance solution of appropriate concentration, and use high performance liquid chromatography to inject and analyze the eluent and sophora flavescens reference substance solution. Results are shown in Figure 3 As shown in A. The results showed that one of the chromatographic peaks in the Sophora flavescens eluate was determined to be matrine. The above results determined that one of the components in Sophora flavescens that can bind to colon cancer exosomes is matrine. Example 5

[0028] Activity inhibition test of matrine on HT-29 colon cancer cells and half inhibitory concentration IC 50 HT-29 colon cancer cells in the logarithmic growth phase were counted and prepared into a concentration of 6×10 4100 μL of cell suspension of 1 μg / mL was added to a 96-well plate and cultured overnight in a 37°C, 5% CO2 cell culture incubator. After the cells adhered to the wall, different concentrations of matrine were added, with gradient concentrations of 0 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM, 128 μM, 256 μM, 512 μM, and 1024 μM. Three replicates were set for each group, and the drug solvent was used as the control group. After 48 h of culture, 10 μL of CCK-8 working solution was added, and the cells were incubated in the cell culture incubator for another 3 h. The OD value was detected at 450 nm and calculated. Figure 4 48 hours IC for matrine 50 Test results. The results show that the IC of matrine to colon cancer cells 50 is 35.30 μM, which is comparable to the IC value of 5-fluoropyrimidine, a positive drug reported in the literature. 50 The difference is 18 μM, which is not much. This suggests that matrine has a good effect of inhibiting the growth of colon cancer cells, that is, matrine has anti-colon cancer activity. Example 6

[0029] Experiment on optimizing the concentration of Chinese herbal medicine extracts. 0.50 mg / mL, 0.75 mg / mL, and 1 mg / mL of Sophora flavescens extract solutions were prepared respectively, and 200 μL of Sophora flavescens extract was added to the EP tubes containing TDEs@SiO2 respectively. After mixing, the mixture was placed in an oscillator at room temperature for 30 minutes, and centrifuged to obtain three supernatants (unadsorbed liquids). Subsequently, 200 μL of methanol was added to the three EP tubes containing the remaining solids respectively, and the mixture was placed in an oscillator for 30 minutes, and centrifuged to obtain the supernatant (eluent). The initial solution, unadsorbed solution, and eluent of Chinese herbal medicine extracts of different concentrations were analyzed by high performance liquid chromatography. The results showed that the concentrations of 0.75 mg / mL and 1 mg / mL of the Chinese medicine extract were too high, and the liquid chromatography peak areas of the unadsorbed liquid and the eluent changed little, making it impossible to analyze whether TDEs@SiO2 had an adsorption effect; while the concentration of 0.50 mg / mL of Sophora flavescens extract was appropriate, and the liquid chromatography peak areas of the unadsorbed liquid and the eluent were quite different, making it easier to analyze the Sophora flavescens components adsorbed by TDEs@SiO2. Example 7

[0030] Experiment on optimizing the co-incubation time of Chinese herbal medicine extracts and TDEs@SiO2. Prepare 0.50 mg / mL of Sophora flavescens extract solution, add 200 μL of Sophora flavescens extract to 5 EP tubes containing TDEs@SiO2, mix and place in a room temperature oscillator for 20, 30, 40, 50, and 60 minutes, centrifuge to obtain 5 supernatants (unadsorbed liquid), then add 200 μL of methanol to the 5 EP tubes containing the remaining solids, mix and place in an oscillator for 30 minutes, centrifuge to obtain the supernatant (elution). The initial solution, the unadsorbed solution at different co-incubation times, and the eluate at different co-incubation times were analyzed by high performance liquid chromatography. The results showed that the peak area of ​​the non-adsorbed liquid after 20 minutes of co-incubation was not much different from that of the initial liquid, and the eluate had no obvious peak area; there was no significant difference in the peak area of ​​the non-adsorbed liquid after 20, 30, 40, 50, and 60 minutes of co-incubation, that is, 30 minutes was selected as the co-incubation time of Chinese medicine extract and TDEs@SiO2. Example 8

[0031] Experiment on time optimization of solution elution and TDEs@SiO2 binding components. Prepare 0.50 mg / mL of Sophora flavescens extract solution, add 200 μL of Sophora flavescens extract to 5 EP tubes containing TDEs@SiO2, mix and place in a room temperature oscillator for 30 minutes, centrifuge to obtain 5 supernatants (non-adsorbed liquid), then add 200 μL of methanol to the 5 EP tubes containing the remaining solid, mix and place in an oscillator for 20, 30, 40, 50, 60 minutes, centrifuge to obtain 5 supernatants (eluents). The initial solution of Chinese medicine extract, non-adsorbed solution, and eluents at different elution times were analyzed by high performance liquid chromatography. The results showed that the components adsorbed on TDEs@SiO2 could not be completely eluted at 20 minutes, while the HPLC peaks were messy at 40, 50, and 60 minutes, which would elute the exosomes in TDEs@SiO2 and damage the TDEs@SiO2 carrier material, so an elution time of 30 minutes was selected.

[0032] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0033] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for screening anti-colon cancer active ingredients, comprising the following steps: (1) Preparation of exosome affinity chromatography carrier material: First, glutaraldehyde and aminosilica were shaken on an oscillator at room temperature for a certain period of time to add aldehyde groups to the silica, centrifuged and filtered, and then colon cancer cell exosomes were added and incubated for a certain period of time to make the aldehyde groups of aminosilica covalently bind to the carboxyl groups of the exosomes, and then lysine was added and shaken for a certain period of time to block the excess aldehyde groups on the silica, thereby obtaining exosome silica (TDEs@SiO2) affinity chromatography carrier material. (2) Screening of anti-colon cancer active ingredients of traditional Chinese medicine: Affinity chromatography was used to separate traditional Chinese medicine ingredients that can bind to cancer cell exosomes. First, the traditional Chinese medicine extract was dissolved into an appropriate concentration and then added to the TDEs@SiO2 affinity chromatography carrier material obtained by (1) according to a certain concentration volume, so that the traditional Chinese medicine extract and the TDEs@SiO2 affinity chromatography carrier material were incubated for a certain period of time, and the unadsorbed supernatant was removed by centrifugation, which was the unadsorbed liquid. The solution bound to TDEs@SiO2 is obtained by eluting with a suitable solvent for a certain period of time, which is the eluent. The initial solution of the Chinese medicine extract, the unadsorbed solution, and the eluent are respectively injected into a high-performance liquid chromatograph for detection. By analyzing the changes in the peak areas of the components in different solutions, the components bound to the Chinese medicine and TDEs@SiO2 are screened out; the components bound to the exosomes are identified by liquid chromatography-mass spectrometry; the half-maximal inhibitory concentration of the screened and identified binding components on colon cancer cells is determined, and finally the active components with anti-colon cancer effects are obtained.

2. The method for screening anti-colon cancer active ingredients according to claim 1, characterized in that: The concentration of the Chinese herbal medicine extract solution is 0.5 mg / mL.

3. The method for screening anti-colon cancer active ingredients according to claim 1, characterized in that: The incubation time of the traditional Chinese medicine extract and the TDEs@SiO2 affinity chromatography carrier material is 30 minutes.

4. The method for screening anti-colon cancer active ingredients according to claim 1, characterized in that: The exosomes are derived from HT-29 colon cancer cells.

5. The method for screening anti-colon cancer active ingredients according to claim 1, characterized in that: The solution combined with TDEs@SiO2 is obtained by eluting with a suitable solvent for a certain period of time, and the certain period of time is 30 minutes.

6. The method for screening anti-colon cancer active ingredients according to claim 1, characterized in that: The affinity chromatography method is specifically as follows: the chromatographic column is YMC-Pack ODS-AC18 (4.6 × 250 mm), the detection wavelength is 254 nm, the mobile phase A is 0.1% phosphoric acid, the mobile phase B is acetonitrile, the gradient elution program is 0 min-95% A; 10 min-75% A; 15 min-65% A; 30min-55% A; 50 min-55% A; 60 min-95% A, the column temperature is 35°C, the wavelength is 220 nm, and the flow rate is 1 mL / min.

7. The method for screening anti-colon cancer active ingredients according to claim 1, characterized in that: The LC-MS method is as follows: the chromatographic column is C18 (3.5 μm; 2.1 mm × 50 mm), the mobile phase A is 0.1% formic acid, the mobile phase B is methanol, and the gradient elution program is 0 min-70% A; 4 min-70% A; 14 min-52% A; 24 min-52% A; 25 min-70% A; 30 min-70% A. The nebulizer pressure is 35 psi, the drying gas flow rate is 8 L / min, the gas temperature is 320°C, the sheath gas temperature is 350°C, the gas flow rate is 11 L / min, and the mass range is selected as 50-3000 (m / z).

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