Affinity chromatographic column for screening active ingredients of traditional Chinese medicine as well as preparation method and application of affinity chromatographic column

Abstract

The invention discloses an affinity chromatographic column for screening active ingredients of traditional Chinese medicines as well as a preparation method and application of the affinity chromatographic column. A 5-hydroxytryptamine 1A receptor protein or a 5-hydroxytryptamine transporter protein is immobilized on a stationary phase of the affinity chromatographic column; according to the affinity chromatographic column disclosed by the invention, 5-hydroxytryptamine 1A receptor protein or 5-hydroxytryptamine transporter protein is fixed on the stationary phase, so that the active ingredients for treating depression in the traditional Chinese medicine extracting solution can be reserved, and the active ingredients can be determined according to the retention time, so that the screening of the active ingredients of the traditional Chinese medicines for treating depression is achieved, and the screening efficiency is improved. A technical basis is provided for traditional Chinese medicine research.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to an affinity chromatography column for screening active ingredients of traditional Chinese medicines, and a preparation method and application thereof. Background Art

[0002] The active ingredients of traditional Chinese medicine are the material basis for its treatment of diseases and an important source for finding new lead compounds for innovative drugs. However, due to the complexity of the composition of traditional Chinese medicine itself, the integrity of its effects and the ambiguity of its mechanism of action, clarifying the effective ingredients of traditional Chinese medicine and their mechanism of action is still a key issue facing traditional Chinese medicine research. In recent years, in the context of integrating relevant knowledge from many disciplines, a series of research ideas and methods for the effective ingredients of traditional Chinese medicine have been developed, mainly including the screening of effective ingredients based on the "spectrum-effect" relationship of traditional Chinese medicine, the screening of effective ingredients based on serum pharmacology and serum pharmacochemistry, the screening of effective ingredients based on targeted fishing and cell membrane chromatography technology, the screening of effective ingredients based on surface plasmon biosensors, the screening of effective ingredients based on fluorescent probe ligand labeling, and the pharmacophore prediction technology based on computer virtual screening.

[0003] The spectrum-effect relationship of traditional Chinese medicine is to construct the fingerprint spectrum of traditional Chinese medicine by using analytical techniques such as chromatography and mass spectrometry, and to conduct qualitative and quantitative analysis of the chemical components in the fingerprint spectrum. In combination with the efficacy and indications of traditional Chinese medicine, appropriate efficacy models are selected to evaluate the efficacy of chemical components, so as to achieve spectrum-effect correlation analysis and establish the "spectrum-effect" relationship of traditional Chinese medicine.

[0004] Serum pharmacology is the study of the pharmacodynamic effects of drug components of traditional Chinese medicine that enter the serum through metabolism in the body, while serum medicinal chemistry is the process of separating and identifying active compounds in serum. Modern multidimensional combined techniques are used to analyze the metabolites / free components in serum. The biological activity is evaluated by in vitro and in vivo pharmacological methods to identify the effective components of traditional Chinese medicine. However, this method also has certain limitations: (1) Due to the complexity of biological samples, the difficulty of separation and analysis is comparable to that of the analysis of traditional Chinese medicine compound prescriptions; (2) It is difficult to infer the structural information of the active components of traditional Chinese medicine from the metabolites of traditional Chinese medicine; (3) Some compounds detected in serum may be inactive components of traditional Chinese medicine; (4) A single compound appearing in serum has significant therapeutic effect only when the blood drug concentration exceeds the original concentration.

[0005] Traditional Chinese medicine needs to bind to the target in the body to exert its efficacy, and this binding requires a certain affinity between the molecule and the protein. Targeted fishing and cell membrane chromatography technology are based on this principle. The technology is to load enzymes, cell membranes or cells on the chromatographic stationary phase, and show differences in retention behavior according to the differences in the affinity of different drug chemical components to the target on the cell membrane, thereby realizing the screening of active ingredients of drugs in complex systems. The essence of this method is the affinity between the drug and the target. However, due to the complex components in the cell membrane of the immobilized high-expression protein and the large non-specific interference, the specificity of the method is poor, and the false positive rate is high in the screening of effective ingredients of traditional Chinese medicine.

[0006] The effective ingredient screening technology based on surface plasmon biosensor is a biosensing analysis technology based on the principle of surface plasmon resonance (SPR). It can directly monitor and track the interactions between various biomolecules in real time by measuring the changes in the physical optical reflectivity of the analyte SPR.

[0007] The effective ingredient screening technology based on fluorescent probe ligand labeling uses specific fluorescent probes to label ligands or targets, and screens the effective ingredients of complex systems by detecting changes in fluorescence intensity or enzyme activity before and after the ligand binds to the target.

[0008] The pharmacophore prediction technology based on computer virtual screening uses database mining and multi-target virtual screening to solve the problems of complex ingredients and unclear mechanisms of action of traditional Chinese medicine. However, although it can process huge amounts of data and comprehensively evaluate the activity of traditional Chinese medicine ingredients based on different algorithms and scoring functions, it is mainly based on predictive results and has a high false positive rate.

[0009] In view of this, the present invention is proposed. Summary of the invention

[0010] The purpose of the present invention is to provide an affinity chromatography column for screening active ingredients of traditional Chinese medicine, a preparation method and an application thereof. The affinity chromatography column of the present invention can realize the screening of active ingredients of traditional Chinese medicine for treating depression, and provide a technical basis for traditional Chinese medicine research.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted: A first aspect of the present invention provides an affinity chromatography column for screening active ingredients of traditional Chinese medicines, wherein a 5-hydroxytryptamine 1A receptor protein or a 5-hydroxytryptamine transporter protein is fixed on the stationary phase of the affinity chromatography column.

[0012] The second aspect of the present invention provides a method for preparing the above affinity chromatography column for screening active ingredients of traditional Chinese medicine, the preparation method comprising the following steps: (a) The 5-HT1A receptor labeled with dehalogenase (Halo) was inserted into the pET15b vector to obtain pET15b-5-HT 1A R-Halo, and then pET15b-5-HT 1A R – Halo transfer in E. coli BL21 (DE3) to obtain the first recombinant strain; Or insert the 5-HT transporter labeled with Halo into the PET15b vector to obtain pET15b-5-HTT-Halo, and then transfer pET15b-5-HTT-Halo into E. coli BL21 (DE3) PlysS , obtaining a second recombinant strain; (b) inoculating the first recombinant strain or the second recombinant strain into an agarose solid medium containing sodium ampicillin for streaking culture, picking a single colony into an LB primary medium containing sodium ampicillin for overnight culture, and then transferring the colony to a composite autoinduction medium containing ampicillin at a ratio of 6% to 10% of the volume of the composite autoinduction medium for culture, and when the OD value reaches 0.4 to 0.6, recovering the bacterial cells, placing the bacterial cells in a bacterial lysis solution for disruption, centrifuging, and collecting the supernatant; (c) under solvent conditions, aminopropyl silica gel, 6-chlorohexanoic acid, HATU, and N,N-diisopropylethylamine are mixed and mechanically stirred at room temperature, and then filtered and washed to obtain 6-chlorohexanoic acid-modified silica gel; (d) The supernatant was mixed with 6-chlorohexanoic acid-modified silica gel and mechanically stirred at room temperature, and then filtered and washed to obtain immobilized 5-HT. 1A R chromatographic stationary phase or immobilized 5-HTT chromatographic stationary phase; (e) Immobilized 5-HT 1A The affinity chromatography column for screening the active ingredients of Yueju Pills is prepared by packing the column with R chromatographic stationary phase or immobilized 5-HTT chromatographic stationary phase.

[0013] Preferably, in the step (b), the content of ampicillin sodium in the agarose solid medium containing ampicillin sodium, the LB primary medium containing ampicillin sodium, and the composite auto-induction medium containing ampicillin is 80-120 μg / mL, respectively.

[0014] Preferably, in step (b), the streak culture temperature is 30-37°C and the time is 12-18h; The culture in LB primary liquid medium was cultured at 30-37°C and 200-250 rpm in a shaking incubator for 10-14 h; The culture in the composite autoinduction medium was carried out at 25-37°C and incubated on a shaking platform at 200-250 rpm.

[0015] Preferably, in step (b), the mass ratio of the bacterial cells to the lysis solution is 1:(8-12); the lysis solution is a mixture of 0.01-0.03M PB, 0.4-0.6M NaCl, and pH 7.2-7.6.

[0016] Preferably, in step (b), the crushing treatment is performed 1 to 3 times, and each crushing treatment is performed in an ice bath with ultrasonic crushing for 8 to 12 seconds at intervals of 12 to 18 seconds, and lasting for 22 to 28 minutes.

[0017] Preferably, in step (c), the mass ratio of aminopropyl silica gel, 6-chlorohexanoic acid, HATU and N,N-diisopropylethylamine is 1:(1-1.5):(1-1.5):(2-4); The solvent is N,N-dimethylformamide.

[0018] Preferably, in the step (d), the mass volume ratio of the 6-chlorohexanoic acid-modified silica gel to the supernatant is 1:(4-8).

[0019] The third aspect of the present invention provides an application of the affinity chromatography column for screening active ingredients of traditional Chinese medicine or the affinity chromatography column for screening active ingredients of traditional Chinese medicine obtained by the above preparation method in screening active ingredients of Yueju Pills.

[0020] A fourth aspect of the present invention provides a method for screening the active ingredients of Yueju Pills, the screening method comprising the following steps: Refluxing the medicinal materials in Yueju Pills with 70% ethanol to obtain an extract; Using the affinity chromatography column for screening active ingredients of traditional Chinese medicine or the affinity chromatography column for screening active ingredients of traditional Chinese medicine obtained by the preparation method to perform chromatographic analysis on the active ingredients in the extract, and then determining the active ingredients based on the chromatographic analysis and mass spectrometry identification results; The mobile phase in the chromatographic analysis is 15-25 mM ammonium acetate with a pH value of 7.2-7.6; flow rate: 0.1-0.3 mL / min; detection wavelength: 254 nm, 330 nm, 340 nm; injection volume: 10-20 μL; column temperature: 22-28 °C.

[0021] Compared with the prior art, the beneficial effects of the present invention include at least: The affinity chromatography column of the present invention can retain the active ingredients of depression by fixing 5-hydroxytryptamine 1A receptor protein or 5-hydroxytryptamine transporter protein on the stationary phase, and the active ingredients can be determined by the difference in retention time, thereby achieving the screening of active ingredients of traditional Chinese medicine for treating depression, providing a technical basis for traditional Chinese medicine research; in addition, through the screening of the active ingredients of Yueju Pills, the antidepressant efficacy ingredients of the prescription are identified as artemisinin, atractylodes and α-cyperus rotundus, which are respectively derived from Chuanxiong, Atractylodes and Cyperus rotundus in the prescription, providing a new technical idea for analyzing the material basis of the efficacy of traditional Chinese medicine, revealing the compatibility mechanism of compound prescriptions and establishing a quality control system for traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0023] Figure 1 This is an SDS-PAGE analysis diagram in Example 1 of the present invention; Figure 2 5-HT in Example 1 of the present invention 1A Specificity investigation diagram of R column and 5-HTT column; Figure 3 The extract of Yueju Pills in Example 2 of the present invention is in 5-HT 1A Chromatograms on R column and 5-HTT column; Figure 4 The extract of Yueju Pills in Example 2 of the present invention is in 5-HT 1A Total ion chromatograms of the retained components on the R column and 5-HTT column; Figure 5 is a primary mass spectrum of ligustilide in the retained component in Example 2 of the present invention; Figure 6 is the secondary mass spectrum of ligustilide in the retained component in Example 2 of the present invention; Figure 7 is the primary mass spectrum of α-cyperone in the retained component in Example 2 of the present invention; Figure 8 is the secondary mass spectrum of α-cyperone in the retained component in Example 2 of the present invention; Fig. 9 This is the primary mass spectrum of atractylodesin in the retained component in Example 2 of the present invention; Fig.10 This is the primary mass spectrum of atractylodesin in the retained component in Example 2 of the present invention. DETAILED DESCRIPTION

[0024] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0025] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0026] The embodiment of the present invention provides an affinity chromatography column for screening active ingredients of traditional Chinese medicines, wherein a 5-hydroxytryptamine 1A receptor protein or a 5-hydroxytryptamine transporter protein is fixed on the stationary phase of the affinity chromatography column.

[0027] The affinity chromatography column of the present invention can retain the active ingredients of depression by fixing the 5-hydroxytryptamine 1A receptor protein or the 5-hydroxytryptamine transporter protein on the stationary phase. The active ingredients can be determined by the retention time, thereby achieving the screening of active ingredients of traditional Chinese medicine for treating depression, providing a technical basis for traditional Chinese medicine research.

[0028] Another embodiment of the present invention provides a method for preparing the above affinity chromatography column for screening active ingredients of traditional Chinese medicine, the preparation method comprising the following steps: (a) The 5-HT1A receptor labeled with Halo was inserted into the pET15b vector to obtain pET15b-5-HT 1A R-Halo, and then pET15b-5-HT 1A R – Halo transfer in E. coli BL21 (DE3) to obtain the first recombinant strain; Or insert the 5-HT transporter labeled with Halo into the PET15b vector to obtain pET15b-5-HTT-Halo, and then transfer pET15b-5-HTT-Halo into E. coli BL21 (DE3) PlysS , obtaining a second recombinant strain; (b) inoculating the first recombinant strain or the second recombinant strain into an agarose solid medium containing sodium ampicillin for streaking culture, picking a single colony into an LB primary medium containing sodium ampicillin for overnight culture, and then transferring the colony to a composite autoinduction medium containing ampicillin at a ratio of 6% to 10% of the volume of the composite autoinduction medium for culture, and when the OD value reaches 0.4 to 0.6, recovering the bacterial cells, placing the bacterial cells in a bacterial lysis solution for disruption, centrifuging, and collecting the supernatant; (c) under solvent conditions, aminopropyl silica gel, 6-chlorohexanoic acid, HATU, and N,N-diisopropylethylamine are mixed and mechanically stirred at room temperature, and then filtered and washed to obtain 6-chlorohexanoic acid-modified silica gel; (d) The supernatant was mixed with 6-chlorohexanoic acid-modified silica gel and mechanically stirred at room temperature, and then filtered and washed to obtain immobilized 5-HT. 1A R chromatographic stationary phase or immobilized 5-HTT chromatographic stationary phase; (e) Immobilized 5-HT 1A The affinity chromatography column for screening the active ingredients of Yueju Pills is prepared by packing the column with R chromatographic stationary phase or immobilized 5-HTT chromatographic stationary phase.

[0029] In some embodiments, in step (b), the content of ampicillin sodium in the agarose solid medium containing ampicillin sodium, the LB primary medium containing ampicillin sodium, and the composite auto-induction medium containing ampicillin can be 80-120 μg / mL, respectively.

[0030] In some embodiments, in step (b), the streak culture temperature may be 30-37° C. and the time may be 12-18 hours; The culture in LB primary liquid medium was cultured at 30-37°C and 200-250 rpm in a shaking incubator for 10-14 h; The culture in the composite autoinduction medium was carried out at 25-37°C and incubated on a shaking platform at 200-250 rpm.

[0031] In some embodiments, in step (b), the mass ratio of the bacteria to the lysis solution may be 1: (8-12); the lysis solution may be a mixture of 0.01-0.03M PB, 0.4-0.6M NaCl, and pH 7.2-7.6.

[0032] In some embodiments, in step (b), the crushing treatment may be performed 1 to 3 times, and each crushing treatment is performed under ice bath conditions, with ultrasonic crushing for 8 to 12 seconds at intervals of 12 to 18 seconds, and lasting for 22 to 28 minutes.

[0033] In some embodiments, in step (c), the mass ratio of aminopropyl silica gel, 6-chlorohexanoic acid, HATU and N,N-diisopropylethylamine is 1: (1-1.5): (1-1.5): (2-4); The solvent is N,N-dimethylformamide.

[0034] In some embodiments, in step (d), the mass volume ratio of the 6-chlorohexanoic acid-modified silica gel to the supernatant can be 1:(4-8).

[0035] Another embodiment of the present invention provides an application of the affinity chromatography column for screening active ingredients of traditional Chinese medicine or the affinity chromatography column for screening active ingredients of traditional Chinese medicine obtained by the above preparation method in screening active ingredients of Yueju Pills.

[0036] Another embodiment of the present invention provides a method for screening the active ingredients of Yueju Pills, the screening method comprising the following steps: Refluxing the medicinal materials in Yueju Pills with 70% ethanol to obtain an extract; Using the affinity chromatography column for screening active ingredients of traditional Chinese medicine or the affinity chromatography column for screening active ingredients of traditional Chinese medicine prepared by the above preparation method to perform chromatographic analysis on the active ingredients in the extract, and then determining the active ingredients according to the chromatographic analysis results; The mobile phase in the chromatographic analysis is 15-25 mM ammonium acetate with a pH value of 7.2-7.6; flow rate: 0.1-0.3 mL / min; detection wavelength: 254 nm, 330 nm, 340 nm; injection volume: 10-20 μL; column temperature: 22-28°C.

[0037] The technical solution of the present invention is further described in detail below through specific embodiments.

[0038] Example 1 This embodiment is a method for preparing an affinity chromatography column for screening active ingredients of traditional Chinese medicine, and the preparation method comprises the following steps: (a) The 5-HT1A receptor (Halo-linked protein C-terminus) labeled with Halo was inserted into the pET15b vector to obtain pET15b-5-HT 1A R-Halo, and then pET15b-5-HT 1A R-Halo is transferred by heat shock E. coli BL21 (DE3) to obtain the first recombinant strain; Alternatively, the 5-HT transporter labeled with Halo (Halo connexin C-terminus) was inserted into the PET15b vector to obtain pET15b-5-HTT-Halo, and then pET15b-5-HTT-Halo was transferred into E. coli BL21 (DE3) PlysS , obtaining a second recombinant strain; (b) Prepare agarose solid culture medium, LB primary culture medium, and composite autoinduction culture medium respectively, sterilize them by wet method, and when the temperature of the sterilizer drops to 60°C, take out the culture medium and place it on the clean bench for use. Prepare ampicillin sodium at a concentration of 100 mg / mL, draw an appropriate amount and add it to the above culture medium until the final antibiotic concentration is 100 μg / mL, shake the agarose solid culture medium and quickly pour it into the plate until it condenses into a solid, inoculate the first recombinant strain or the second recombinant strain into the agarose solid culture medium containing ampicillin sodium for streak culture, and culture at 37°C for 15 hours; pick a single colony to LB primary culture medium containing ampicillin sodium for overnight culture, and the culture conditions are 37°C and 220 rpm shaking incubator; then, transfer it to the composite autoinduction culture medium containing ampicillin at a ratio of 8% of the volume of the composite autoinduction culture medium for culture, and the culture conditions are 30°C and 220 rpm shaking incubator. When the OD value reaches 0.5, recover the bacteria, and place the bacteria in the lysis solution (the lysis solution is 0.02 mol / LPB, 0.5 mol / L NaCl, pH 7.3) according to the ratio of the wet weight of the bacteria to the lysis solution of 1:10. 7.4) Perform ultrasonic disruption in an ice-water bath for 10 s, 15 s interval, 25 min, a total of 2 disruptions, centrifuge, and collect the supernatant; (c) Using N,N-dimethylformamide (DMF) as solvent, add aminopropyl silica gel (1.0 eq), 6-chlorohexanoic acid (1.2 eq), HATU (1.2 eq), and N,N-diisopropylethylamine (DIEA, 3.0 eq) in the following ratios, stir mechanically at room temperature for 4 h, filter using a G4 vertical melting funnel, wash with DMF, methanol, and 20 mmol / L PB solution three times in sequence, and obtain 6-chlorohexanoic acid-modified silica gel; (d) The supernatant and the 6-chlorohexanoic acid-modified silica gel were mixed at a mass volume ratio of 1:6 and mechanically stirred at room temperature for 1.5 h. The mixture was filtered using a G4 vertical melting funnel, and the filter cake was washed three times with 20 mmol / L PB to obtain immobilized 5-HT. 1A R chromatographic stationary phase or immobilized 5-HTT chromatographic stationary phase, the stationary phase was stored in 20 mmol / L PB at 4°C; (e) Immobilized 5-HT 1A R chromatographic stationary phase or immobilized 5-HTT chromatographic stationary phase was wet packed, the column standard was 4.6 mm × 30 mm, 6 μm; 20 mmol / L PB was selected as the displacement liquid and homogenate, 400 bar pressure, and the column was packed in an ice bath for 40 min to obtain the affinity chromatography column for screening the active ingredients of Yueju Pills (respectively denoted as 5-HT 1A R column or 5-HTT column).

[0039] 1. Perform gel electrophoresis on the bacterial cells recovered from LB primary medium, the bacterial cells recovered from the induction medium, and the supernatant and precipitate of the cell lysis in the free island medium; the electrophoresis results are as follows: Figure 1 As shown, Figure 1 In the middle, Halo tag is fused with 5-HT 1A R (A), Halo tag fusion 5-HTT (B); Depend on Figure 1 It can be seen that: Halo tag fused to 5-HT 1A There is a particularly obvious band between 66.2 kDa and 116.0 kDa of the standard protein molecular weight in the autoinduction culture medium and cell lysate supernatant of R and 5-HTT recombinant protein. The band is almost invisible in the cell lysate precipitate of the autoinduction culture medium. After one-step regression calculation, the molecular weights are approximately 80.0 kDa and 104.3 kDa, respectively. It is identified that the band is the induced expression of Halo tag fusion 5-HT 1A R and 5-HTT recombinant proteins.

[0040] 2. The 5-HT prepared above 1A Characterize the specificity of the R column or 5-HTT column: The prepared 5-HT was analyzed by Shimadzu HPLC system. 1A The chromatographic retention behavior of the R column or 5-HTT column was characterized, and sodium nitrite, zolmitriptan, 5-hydroxytryptamine, and buspirone solutions were prepared to characterize the 5-HT 1A R column specificity, the flow rate was set to 0.2 mL / min, and the detection wavelengths were 254 nm, 283 nm, 275 nm and 250 nm, respectively. Sodium nitrite, paroxetine, sertraline, citalopram and fluoxetine solutions were prepared to characterize the specificity of 5-HTT column, the flow rate was set to 0.6 mL / min, and the detection wavelengths were 254 nm, 295 nm, 265 nm, 238 nm and 227 nm, respectively. Ammonium acetate (pH 7.4) with a concentration of 20 mmol / L was used as the mobile phase. Sodium nitrite was a non-retained component, and its retention time could be measured to determine the dead time t0 of the chromatographic system.

[0041] Under the same chromatographic conditions, the effects of zolmitriptan, 5-hydroxytryptamine, and buspirone on the 5-HT 1A The retention behavior of paroxetine, sertraline, citalopram and fluoxetine on the 5-HTT column was used to characterize the column specificity. The dead time (t0) of the system was determined by the retention behavior of sodium nitrite. The retention behavior of the ligand and sodium nitrite was shown in Figure 2 As shown. It can be seen that different ligands have different effects on the immobilized 5-HT 1AThe retention times on the chromatographic columns of 5-HTT and 5-HTT are different, indicating that the immobilized protein has ligand recognition specificity, and there is a certain tailing phenomenon in the chromatographic peak. This is because the binding of the ligand to the immobilized protein is a fast binding and slow dissociation process, so there is a tailing phenomenon. Table 1 shows the determination results of retention time tR and capacity factor k'. The retention times of the ligands of the three proteins on the corresponding chromatographic columns are all greater than the system dead time and the retention time differences are obvious. This shows that after immobilization, 5-HT 1A R and 5-HTT still have biological activity to recognize their specific ligands.

[0042] Table 1 Chromatographic retention behavior of ligands on three chromatographic models ; Example

[0043] This embodiment is a method for screening the active ingredients of Yueju Pills, and the screening method comprises the following steps: 1. Preparation of Yueju Pills Extract: Accurately weigh 50 g of Cyperus rotundus, Ligusticum chuanxiong, Atractylodes lancea (fried), Liushenqu (fried), and Gardenia jasminoides in a weight ratio of 1:1:1:1:1, crush and sieve to obtain coarse powder, add 12 times the amount of 70% ethanol, extract for 1.5 h, extract 3 times, filter the decoction, combine the filtrate, concentrate, and dry to a thick paste to obtain a coarse powder extract of the four herbs, add the fried Gardenia jasminoides fine powder to obtain the extract with the new process prescription.

[0044] Using 5-HT 1A R column and 5-HTT column were used to screen the active ingredients in the above extracts. Chromatographic conditions: 5-HT 1A R column and 5-HTT column (4.6×30 mm, 6 μm); mobile phase: 20 mM ammonium acetate (pH=7.4); flow rate: 0.2 mL / min; detection wavelength: 254 nm, 330 nm, 340 nm; injection volume: 20 μL; column temperature: 25 °C, and the retained chromatographic peaks were collected for further analysis; Vinegar-scented Cyperus rotundus, Chuanxiong, stir-fried Gardenia jasminoides, stir-fried Atractylodes macrocephala, and stir-fried Liushenqu were all purchased from Beijing Tong Ren Tang Pharmacy. They are mainly used to treat six depressions. Modern pharmacological studies have shown that the drug has an antidepressant effect. 5-HT 1A R and 5-HTT are the main targets of drug efficacy. Based on this, it can be inferred that some active ingredients in "Xiangzhi Jieyu Fang" may interact with 5-HT 1A R and 5-HTT specifically bind. 1A R column and 5-HTT column were used to screen the active ingredients in the extract of "Xiangzhi Jieyu Fang". The results are shown in Figure 3 , Figure 3 Middle A: 5-HT 1A R column, B: 5-HTT column; Xiangzhi Jieyu Fang extract was purified by 5-HT 1A The main chromatographic peaks on the R column and the 5-HTT column were analyzed. According to the dead time analysis of the chromatographic system, the retention time of peak 1 was close to the dead time, so this peak was considered to be an unretained component, that is, it did not contain 5-HT. 1A The retention time of peaks 2, 3, and 4 is significantly greater than the dead time of the chromatographic system, which means that peaks 2, 3, and 4 may contain components that specifically bind to immobilized 5-HT 1A R and 5-HTT specifically bind to each other. Therefore, peaks 2, 3, and 4 were collected to further analyze and identify the components contained therein; Figure 4 The extract of Yueju Pills in 5-HT 1A The total ion chromatogram of the retained components on the R column and the 5-HTT column showed three molecular ion peaks at 1.16 min, 2.82 min and 3.41 min in positive ion mode, with m / z 191.7, m / z 219.17 and m / z 183.08 ( Figure 5 , 7 , 9), indicating that the extract contains three 1A R and 5-HTT protein binding components. Secondary mass spectrometry fragmentation analysis was performed using m / z 191.7, m / z 219.17 and m / z 183.08 as parent ions, respectively. Figure 6 , 8 , 10, identified the above three can be combined with 5-HT 1A The components that bind to R and 5-HTT protein are ligustilide, α-cyperone and atractylodesin. Ligustilide comes from Chuanxiong in the original formula, α-cyperone comes from Cyperus rotundus, and atractylodesin comes from Atractylodes lancea.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. An affinity chromatography column for screening active ingredients of traditional Chinese medicine, characterized in that: The stationary phase of the affinity chromatography column is fixed with 5-hydroxytryptamine 1A receptor protein or 5-hydroxytryptamine transporter protein.

2. The method for preparing the affinity chromatography column for screening active ingredients of traditional Chinese medicine according to claim 1, characterized in that: The preparation method comprises the following steps: (a) Insert the 5-HT1A receptor labeled with dehalogenase into the pET15b vector to obtain pET15b-5-HT 1A R-Halo, and then pET15b-5-HT 1A R – Halo transfer in E. coli In BL21 (DE3), the first recombinant strain was obtained; Or insert the 5-HT transporter labeled with Halo into the PET15b vector to obtain pET15b-5-HTT-Halo, and then transfer pET15b-5-HTT-Halo into E. coli BL21 (DE3) PlysS , obtaining a second recombinant strain; (b) inoculating the first recombinant strain or the second recombinant strain into an agarose solid medium containing sodium ampicillin for streaking culture, picking a single colony into an LB primary medium containing sodium ampicillin for overnight culture, and then transferring the colony to a composite autoinduction medium containing ampicillin at a ratio of 6% to 10% of the volume of the composite autoinduction medium for culture, and when the OD value reaches 0.4 to 0.6, recovering the bacterial cells, placing the bacterial cells in a bacterial lysis solution for disruption, centrifuging, and collecting the supernatant; (c) under solvent conditions, aminopropyl silica gel, 6-chlorohexanoic acid, HATU, and N,N-diisopropylethylamine are mixed and mechanically stirred at room temperature, and then filtered and washed to obtain 6-chlorohexanoic acid-modified silica gel; (d) The supernatant was mixed with 6-chlorohexanoic acid-modified silica gel and mechanically stirred at room temperature, and then filtered and washed to obtain immobilized 5-HT. 1A R chromatographic stationary phase or immobilized 5-HTT chromatographic stationary phase; (e) Immobilized 5-HT 1A The affinity chromatography column for screening the active ingredients of Yueju Pills is prepared by packing the column with R chromatographic stationary phase or immobilized 5-HTT chromatographic stationary phase.

3. The preparation method according to claim 2, characterized in that: In the step (b), the contents of ampicillin sodium in the agarose solid culture medium containing ampicillin sodium, the LB primary culture medium containing ampicillin sodium, and the composite auto-induction culture medium containing ampicillin sodium are 80-120 μg / mL, respectively.

4. The preparation method according to claim 1, characterized in that: In the step (b), the streak culture temperature is 30-37°C and the time is 12-18 hours; The culture in LB primary liquid medium was cultured at 30-37°C and 200-250 rpm in a shaking incubator for 10-14 h; The culture in the composite autoinduction medium was carried out at 25-37°C and incubated on a shaking platform at 200-250 rpm.

5. The preparation method according to claim 1, characterized in that: In the step (b), the mass ratio of the bacterial cells to the lysis solution is 1: (8-12); the lysis solution is a mixture of 0.01-0.03M PB, 0.4-0.6M NaCl, and pH 7.2-7.

6.

6. The preparation method according to claim 1, characterized in that: In the step (b), the crushing treatment is performed 1 to 3 times, and each crushing treatment is performed under ice bath conditions, with ultrasonic crushing for 8 to 12 seconds at intervals of 12 to 18 seconds, and lasting for 22 to 28 minutes.

7. The preparation method according to claim 1, characterized in that: In the step (c), the mass ratio of aminopropyl silica gel, 6-chlorohexanoic acid, HATU and N,N-diisopropylethylamine is 1: (1-1.5): (1-1.5): (2-4); The solvent is N,N-dimethylformamide.

8. The preparation method according to claim 1, characterized in that: In the step (d), the mass volume ratio of the 6-chlorohexanoic acid modified silica gel to the supernatant is 1:(4-8).

9. Use of the affinity chromatography column for screening active ingredients of traditional Chinese medicines according to claim 1 or the affinity chromatography column for screening active ingredients of traditional Chinese medicines prepared by any one of the preparation methods of claims 2 to 8 in screening active ingredients of Yueju Pills.

10. A method for screening active ingredients of Yueju Pills, characterized in that: The screening method comprises the following steps: Refluxing the medicinal materials in Yueju Pills with 70% ethanol to obtain an extract; Using the affinity chromatography column for screening active ingredients of traditional Chinese medicines according to claim 1 or the affinity chromatography column for screening active ingredients of traditional Chinese medicines prepared by any one of the preparation methods of claims 2 to 8 to perform chromatographic analysis on the active ingredients in the extract, and then determining the active ingredients according to the chromatographic analysis results; The mobile phase in the chromatographic analysis is 15-25 mM ammonium acetate with a pH value of 7.2-7.6; flow rate: 0.1-0.3 mL / min; detection wavelength: 254 nm, 330 nm, 340 nm; injection volume: 10-20 μL; column temperature: 22-28 °C.

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