Screening method for active ingredients of Gushukang preparation based on cell membrane chromatography technology

Abstract

The invention provides a screening method for active ingredients of a Gushukang preparation based on a cell membrane chromatography technology, and belongs to the technical field of traditional Chinese medicine substance screening. The method comprises the following steps: S1, preparing an osteoblast cell membrane chromatographic column, S2, treating a Gushukang extract, S3, performing HPLC-MS / MS analysis, and S4, verifying binding activity. The UMR-106 osteoblast cell membrane is used as a stationary phase, so that the screening process can be directly combined and recognized based on the native conformation of a cell membrane receptor, and cell membrane protein with higher activity is obtained through low-temperature PBS washing, ice-bath ultrasonic cracking and differential centrifugal extraction; four key active components, including icariin, psoralen, pinoresinol diglucoside and ferulic acid, which are specifically bound with osteogenic cell membranes in the Gushukang capsules / granules are successfully screened by adopting a cell membrane chromatography technology, and the screening efficiency and accuracy are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine material screening technology, and more specifically, to a method for screening active ingredients in bone-strengthening preparations based on cell membrane chromatography technology. Background Technology

[0002] Osteoporosis (OP) is a systemic metabolic bone disease characterized by decreased bone mass and destruction of bone microstructure. With the acceleration of global aging, its incidence is showing a significant upward trend. Traditional treatments such as bisphosphonates and estrogen replacement therapy can slow down bone loss, but they have limitations such as gastrointestinal reactions and cardiovascular risks. The traditional Chinese medicine compound preparation, Bone-Soothing Kang capsules / granules, is composed of liver- and kidney-tonifying herbs such as Epimedium, Rehmannia glutinosa, and Astragalus membranaceus. Clinical studies have shown that it improves bone metabolism through multi-target regulation. However, the specific components in the complex composition that exert anti-osteoporosis effects have not yet been identified. Therefore, a screening method for the active ingredients of Bone-Soothing Kang preparation based on cell membrane chromatography is proposed to address this issue. Summary of the Invention

[0003] To overcome the above deficiencies, the present invention provides a method for screening active ingredients of osteoporosis preparations based on cell membrane chromatography to overcome or at least partially solve the above technical problems.

[0004] This invention is implemented as follows:

[0005] This invention provides a method for screening active ingredients in osteoporosis remedies based on cell membrane chromatography, comprising:

[0006] S1. Preparation of osteoblast membrane chromatography column

[0007] Logarithmic growth phase rat osteoblasts were taken, washed three times with PBS pre-cooled to 4°C, and then lysis buffer containing protease inhibitors was added. The cells were then sonicated and lysed in an ultrasonic homogenizer under ice bath conditions. The lysate was centrifuged at 4°C to collect the supernatant, and the membrane proteins were further purified by differential centrifugation.

[0008] Membrane proteins were mixed with activated silica gel in a certain proportion, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added as crosslinking agents. The mixture was coupled by shaking at 4°C for 12 hours. The coupling product was filtered through a 0.22 μm filter membrane and then packed into a bone cell membrane chromatography column to avoid the generation of air bubbles.

[0009] S2, osteoporosis extract processing

[0010] The contents of the bone-strengthening capsules were placed in methanol and extracted by ultrasonic cleaning. After centrifugation, the supernatant was collected and dried under nitrogen. The residue was dissolved in 10% DMSO-PBS solution and filtered through a 0.22 μm filter membrane. 200 μL of the sample was loaded onto an osteoblast membrane chromatography column at a flow rate of 10 μL / min. After equilibration, the sample was eluted according to the set gradient elution program. The fraction bound to the chromatographic column was collected and lyophilized for storage.

[0011] S3, HPLC-MS / MS analysis

[0012] A C18 column was used at a column temperature of 30℃. 0.1% formic acid water was used as mobile phase A and acetonitrile was used as mobile phase B. Gradient elution was performed at a flow rate of 1.0 mL / min. The injection volume was 10 μL. Each elution peak was collected and the components were identified by mass spectrometry.

[0013] S4, Combined with activity verification

[0014] A competitive binding experiment was conducted by adding standard to the osteoporosis extract. The chromatographic loading steps were repeated, the changes in chromatographic peak area were recorded, and the binding rate was calculated: Binding rate (%) = (1 - peak area of ​​the competitive group / peak area of ​​the control group) × 100;

[0015] The effect of the binding components on the proliferation of UMR-106 osteoblasts was further detected by the MTT assay, alkaline phosphatase activity was detected by the ALP kit, and the formation of mineralized nodules was observed by alizarin red staining.

[0016] In a preferred embodiment, the rat osteoblasts in S1 are the UMR-106 cell line, and the PBS is phosphate-buffered saline with a pH of 7.4.

[0017] In a preferred embodiment, the lysis buffer in S1 consists of 50 mM Tris-HCl and 1 mM EDTA.

[0018] In a preferred embodiment, the ultrasonic crushing in S1 uses a KJ-200 model crusher with a power of 200W and an ultrasonic pulse mode of 5 seconds of operation followed by a 10-second interval.

[0019] In a preferred embodiment, the activated silica gel in S1 has a particle size of 5 μm and a pore size of [missing information]. The membrane protein was then mixed with activated silica gel at a ratio of 1:10.

[0020] In a preferred embodiment, the ultrasonic cleaner in S2 is a model JY92-200 with a frequency of 40kHz, a power of 200W, and an oscillation time of 30 minutes.

[0021] In a preferred embodiment, the gradient elution in S2 is performed as follows: 0-5 min with 100% PBS as the mobile phase; 5-15 min with methanol as the mobile phase, wherein the methanol concentration increases from 0% to 30%; and 15-20 min with methanol as the mobile phase, wherein the methanol concentration increases from 30% to 50%.

[0022] In a preferred embodiment, the C18 column in S3 has a length of 250 mm, an inner diameter of 4.6 mm, and a packing particle size of 5 μm.

[0023] In a preferred embodiment, the gradient elution in S3 is performed such that mobile phase B is linearly increased from 5% to 30% during the period of 0–10 minutes; and linearly increased from 30% to 60% during the period of 10–20 minutes.

[0024] In a preferred embodiment, the standard in S4 is icariin, psoralen, pinoresinol diglucoside, and ferulic acid.

[0025] This invention provides a method for screening active ingredients in osteoporosis treatment preparations based on cell membrane chromatography, the beneficial effects of which include:

[0026] By using UMR-106 osteoblast membrane as the stationary phase, the screening process can directly identify and bind to the native conformation of cell membrane receptors. Higher-activity cell membrane proteins are obtained through low-temperature PBS washing, ice-bath ultrasonic lysis, and differential centrifugation. EDC / NHS coupling is completed at 4℃, effectively maintaining the integrity of the membrane protein structure and function, and improving the binding efficiency of the immobilized cell membrane chromatography column for the active components in Gushukang. The bound components separated by membrane chromatography are further analyzed by HPLC-MS / MS, enabling rapid and accurate structural identification of the bound components and improving the resolution of trace active substances in traditional Chinese medicine compound formulas. Therefore, cell membrane chromatography technology successfully screened four key active components in Gushukang capsules / granules that specifically bind to osteoblast membranes, including icariin, psoralen, pinoresinol diglucoside, and ferulic acid, significantly improving screening efficiency and accuracy. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1, referring to Figure 1 This invention provides a technical solution: a method for screening active ingredients in bone-strengthening preparations based on cell membrane chromatography, comprising:

[0031] S1. Preparation of osteoblast membrane chromatography column

[0032] Logarithmic growth phase rat osteoblasts were taken, washed three times with PBS pre-cooled to 4°C, and then lysis buffer containing protease inhibitors was added. The cells were then sonicated and lysed in an ultrasonic homogenizer under ice bath conditions. The lysate was centrifuged at 4°C to collect the supernatant, and the membrane proteins were further purified by differential centrifugation.

[0033] Membrane proteins were mixed with activated silica gel in a certain proportion, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added as crosslinking agents. The mixture was coupled by shaking at 4°C for 12 hours. The coupling product was filtered through a 0.22 μm filter membrane and then packed into a bone cell membrane chromatography column to avoid the generation of air bubbles.

[0034] S2, osteoporosis extract processing

[0035] The contents of the bone-strengthening capsules were placed in methanol and extracted using an ultrasonic cleaner. After centrifugation, the supernatant was collected and dried under nitrogen. The residue was dissolved in 10% DMSO-PBS solution and filtered through a 0.22 μm filter membrane. 200 μL of the sample was loaded onto an osteoblast membrane chromatography column at a flow rate of 10 μL / min. After equilibration, elution was performed according to the set gradient elution program, and the components bound to the chromatographic column were collected and lyophilized. The residue was a mixture of dried solid active ingredients remaining in the methanol extract of bone-strengthening capsules after nitrogen drying.

[0036] S3, HPLC-MS / MS analysis

[0037] A C18 column was used at a column temperature of 30℃. 0.1% formic acid water was used as mobile phase A and acetonitrile was used as mobile phase B. Gradient elution was performed at a flow rate of 1.0 mL / min. The injection volume was 10 μL. Each elution peak was collected and the components were identified by mass spectrometry.

[0038] S4, Combined with activity verification

[0039] A competitive binding experiment was conducted by adding standard to the osteoporosis extract. The chromatographic loading steps were repeated, the changes in chromatographic peak area were recorded, and the binding rate was calculated: Binding rate (%) = (1 - peak area of ​​the competitive group / peak area of ​​the control group) × 100;

[0040] The effect of the binding components on the proliferation of UMR-106 osteoblasts was further detected by the MTT assay, alkaline phosphatase activity was detected by the ALP kit, and the formation of mineralized nodules was observed by alizarin red staining.

[0041] In this embodiment, the present invention uses UMR-106 osteoblast membrane as the stationary phase, enabling the screening process to directly identify and bind to cell membrane receptors based on their natural conformation. Compared with traditional chemical separation methods, this approach more closely resembles the actual interaction mode between drugs and target cells, significantly improving the targeting and reliability of active ingredient screening. Cell membrane proteins with higher activity are obtained through low-temperature PBS washing, ice-bath ultrasonic lysis, and differential centrifugation extraction. EDC / NHS coupling is then completed at 4°C, effectively maintaining the integrity of the membrane protein structure and function. This improves the binding efficiency of the immobilized cell membrane chromatography column for the active components in the bone-strengthening agent. The present invention simplifies the complex traditional Chinese medicine system by screening bound components through membrane chromatography and then freeze-drying them, enriching them with bone-related active substances. This lays the foundation for subsequent structural identification and efficacy verification, significantly improving screening efficiency and accuracy.

[0042] The bound components separated by membrane chromatography were further analyzed by HPLC-MS / MS, which enabled rapid and accurate structural identification of the bound components, improving the ability to resolve trace active substances in traditional Chinese medicine compound formulas. Combined with MTT and ALP activity detection and alizarin red mineralization experiments, the promoting effects of the screened components on osteoblast proliferation, differentiation and mineralization can be verified, realizing a complete system from "screening-identification-verification" to ensure that the screened components have actual biological activity and pharmacological significance.

[0043] Example 2 is an explanation of Example 1; please refer to it. Figure 1 Specifically, the rat osteoblasts in S1 are UMR-106 cell lines, and the PBS is phosphate-buffered saline with a pH of 7.4.

[0044] In this embodiment, the UMR-106 osteoblast cell line was used. This cell line has a well-defined origin, stable biological characteristics, and a typical osteoblast-like cell phenotype, capable of continuously expressing membrane proteins and receptors related to osteogenic differentiation. Using this cell line can effectively improve the stability and reproducibility of membrane proteins immobilized on membrane chromatography columns, thereby enhancing the reliability of screening results. Pre-cooling and washing the cells with PBS (phosphate-buffered saline) at pH 7.4 provides an isotonic and stable buffer system close to the physiological environment of osteoblasts, which helps protect the cell membrane structure and membrane protein activity, preventing structural damage to membrane proteins due to pH fluctuations or osmotic pressure changes, thereby improving the targeted binding ability of the subsequent chromatography column.

[0045] Example 3 is an explanation of Example 1; please refer to the provided text. Figure 1 Specifically, the lysis buffer in S1 consists of 50 mM Tris-HCl and 1 mM EDTA.

[0046] In this embodiment, Tris-HCl (50 mM) is a commonly used and stable biochemical buffer that can provide a stable pH environment during cell lysis, reducing the damage to osteoblast membrane protein conformation caused by pH fluctuations, thereby effectively maintaining the native structure and activity of membrane proteins and facilitating subsequent cell membrane immobilization. EDTA (1 mM) can chelate Ca2+. 2+ Divalent metal ions such as Mg2+ inhibit the activity of metal ion-dependent proteases, reduce the hydrolysis of membrane proteins by proteases during lysis, significantly improve the integrity and stability of membrane protein extraction, and are beneficial to improving the quality of membrane chromatography columns.

[0047] Example 4 is an explanation of Example 1; please refer to the provided text. Figure 1 Specifically, in S1, the ultrasonic crushing uses a KJ-200 model crusher with a power of 200W and an ultrasonic pulse mode of 5 seconds of operation followed by a 10-second interval.

[0048] In this embodiment, a KJ-200 ultrasonic disruptor with a power of 200W is used, which can provide sufficient energy to disrupt the cell membrane, achieve rapid and effective osteoblast lysis, thereby releasing a large number of membrane proteins, improving the efficiency and yield of membrane protein extraction. The ultrasonic pulse mode (5s working, 10s interval) can avoid the generation of excessive heat from continuous ultrasound, reduce the inactivation of membrane proteins due to thermal denaturation or structural damage, and facilitate subsequent column immobilization and affinity recognition of active ingredients.

[0049] Example 5 is an explanation of Example 1; please refer to it. Figure 1 Specifically, the activated silica gel in S1 has a particle size of 5 μm and a pore size of... The membrane protein was then mixed with activated silica gel at a ratio of 1:10.

[0050] In this embodiment, a particle size of 5μm and a pore size of Activated silica gel provides a large specific surface area and suitable pore size, enabling membrane proteins to be uniformly adsorbed and immobilized, improving the binding capacity and stability of membrane proteins. Reasonable particle size and pore size can reduce spatial compression or deformation of membrane proteins during coupling, maintain the native conformation and activity of proteins, and benefit the specific binding function of subsequent chromatographic columns.

[0051] Example 6 is an explanation of Example 1; please refer to it. Figure 1 Specifically, the ultrasonic cleaner in S2 is model JY92-200, with a frequency of 40kHz, a power of 200W, and an oscillation time of 30 minutes.

[0052] In this embodiment, a JY92-200 ultrasonic cleaner with a frequency of 40kHz and a power of 200W was used, and the mixture was vibrated for 30 minutes. This process can fully destroy the cell walls or granular structure of the active ingredients in the bone-strengthening capsules, accelerate solvent penetration, and allow the active ingredients of the traditional Chinese medicine to dissolve more completely in methanol, thereby improving the extraction efficiency. Compared with high-temperature reflux or long-term heating extraction, the use of ultrasonic cleaner for gentle vibration extraction can reduce the degradation of heat-sensitive components, ensure the integrity of the chemical structure of the active ingredients, and improve the quality of the extract.

[0053] Example 7 is an explanation of Example 1; please refer to it. Figure 1 Specifically, the gradient elution method in S2 is as follows: 0-5 min with 100% PBS as the mobile phase; 5-15 min with methanol as the mobile phase for linear gradient elution, wherein the methanol concentration increases from 0% to 30%; 15-20 min with methanol as the mobile phase for linear gradient elution, wherein the methanol concentration increases from 30% to 50%.

[0054] In this embodiment, a phased gradient elution (0–5 min 100% PBS, 5–15 min 0–30% methanol, 15–20 min 30–50% methanol) can sequentially elute compounds with different binding abilities to osteoblast membranes. This allows multiple active ingredients in the osteoporosis elution product to be effectively separated based on their affinity, improving separation selectivity. The initial elution with 100% PBS removes non-specifically bound or weakly bound components, providing clear binding peaks for subsequent methanol gradient elution. This avoids weakly bound compounds affecting the screening and identification of active ingredients. Furthermore, the gradient elution uses a gentle solvent increment method, which avoids the loss or denaturation of active ingredients caused by a single wash with strong organic solvents, ensuring the structural integrity of the screened components and facilitating subsequent HPLC-MS / MS identification and biological verification.

[0055] Example 8 is an explanation of Example 1; please refer to it. Figure 1 Specifically, the C18 column in S3 has a length of 250 mm, an inner diameter of 4.6 mm, and a packing particle size of 5 μm.

[0056] In this embodiment, the chromatographic column is 250 mm long and the packing particle size is 5 μm, which can provide a longer theoretical plate number and a larger separation efficiency, so that the complex compounds in the osteoporosis extract can be separated more fully. The conventional column diameter of 4.6 mm ensures sufficient sample capacity and maintains reasonable column efficiency, which is conducive to the accurate detection and identification of trace active ingredients by HPLC-MS / MS and improves analytical sensitivity.

[0057] Example 9, this example is an explanation of Example 1, please refer to it. Figure 1 Specifically, in S3, the gradient elution method is as follows: during the period of 0–10 minutes, the mobile phase B is linearly increased from 5% to 30%; during the period of 10–20 minutes, the mobile phase B is linearly increased from 30% to 60%.

[0058] In this embodiment, segmented linear gradient elution allows for sequential elution based on the polarity differences of each component in the bone susceptibility extract, ensuring thorough separation of various active ingredients and improving component resolution efficiency. The gradient elution method enables weakly and strongly retained compounds to elute at appropriate times, avoiding peak overlap and improving the sensitivity and accuracy of HPLC-MS / MS for detecting trace active ingredients. The linearly increasing elution method is gentler than a single high-proportion organic phase wash, preventing degradation or loss of heat-sensitive or solvent-sensitive components and ensuring the structural integrity of the screened components.

[0059] Example 10: This example is an explanation of Example 1. Please refer to the provided text. Figure 1 Specifically, the standard in S4 is icariin, psoralen, pinoresinol diglucoside and ferulic acid.

[0060] In this embodiment, icariin, psoralen, pinoresinol diglucoside, and ferulic acid were selected as standards. These standards can specifically compete with the active ingredients in the osteoporosis extract, accurately verify the affinity of each binding component to the osteoblast membrane, and improve the reliability of the screening results. The standards are all known osteogenic active compounds. By comparing the changes in chromatographic peaks, the binding rate of the active ingredients can be quantified, providing a quantitative basis for subsequent screening and functional verification.

Claims

1. A method for screening active ingredients in osteoporosis treatment preparations based on cell membrane chromatography, characterized in that, include: S1. Preparation of osteoblast membrane chromatography column; Logarithmic growth phase rat osteoblasts were taken, washed three times with PBS pre-cooled to 4°C, and then lysis buffer containing protease inhibitors was added. The cells were then sonicated and lysed in an ultrasonic homogenizer under ice bath conditions. The lysate was centrifuged at 4°C to collect the supernatant, and the membrane proteins were further purified by differential centrifugation. Membrane proteins were mixed with activated silica gel in a certain proportion, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added as crosslinking agents. The mixture was coupled by shaking at 4°C for 12 hours. The coupling product was filtered through a 0.22 μm filter membrane and then packed into a bone cell membrane chromatography column. S2, processed with osteoporosis extract; The contents of the bone-strengthening capsules were placed in methanol and extracted by ultrasonic cleaning. After centrifugation, the supernatant was collected and dried under nitrogen. The residue was dissolved in 10% DMSO-PBS solution and filtered through a 0.22 μm filter membrane. 200 μL of the sample was loaded onto an osteoblast membrane chromatography column at a flow rate of 10 μL / min. After equilibration, the sample was eluted according to the set gradient elution program. The fraction bound to the chromatographic column was collected and lyophilized for storage. S3, HPLC-MS / MS analysis; A C18 column was used at a column temperature of 30℃. 0.1% formic acid water was used as mobile phase A and acetonitrile was used as mobile phase B. Gradient elution was performed at a flow rate of 1.0 mL / min. The injection volume was 10 μL. Each elution peak was collected and the components were identified by mass spectrometry. S4. Combine with activity verification; A competitive binding experiment was conducted by adding standard to the osteoporosis extract. The chromatographic loading steps were repeated, the changes in chromatographic peak area were recorded, and the binding rate was calculated: Binding rate (%) = (1 - peak area of ​​the competitive group / peak area of ​​the control group) × 100; The effect of the binding components on the proliferation of UMR-106 osteoblasts was further detected by the MTT assay, alkaline phosphatase activity was detected by the ALP kit, and the formation of mineralized nodules was observed by alizarin red staining.

2. The method for screening active ingredients in a bone-strengthening preparation based on cell membrane chromatography according to claim 1, characterized in that, The rat osteoblasts in S1 are the UMR-106 cell line, and the PBS is phosphate-buffered saline with a pH of 7.

4.

3. The method for screening active ingredients in a bone-strengthening preparation based on cell membrane chromatography according to claim 2, characterized in that, The lysis buffer in S1 consists of 50 mM Tris-HCl and 1 mM EDTA.

4. The method for screening active ingredients in a bone-strengthening preparation based on cell membrane chromatography according to claim 3, characterized in that, The ultrasonic crushing in S1 uses a KJ-200 model crusher with a power of 200W and an ultrasonic pulse mode of 5 seconds of operation followed by a 10-second interval.

5. The method for screening active ingredients in a bone-strengthening preparation based on cell membrane chromatography according to claim 4, characterized in that, The activated silica gel in S1 has a particle size of 5μm and a pore size of... The membrane protein was then mixed with activated silica gel at a ratio of 1:

10.

6. The method for screening active ingredients in a bone-strengthening preparation based on cell membrane chromatography according to claim 5, characterized in that, The ultrasonic cleaner in S2 is model JY92-200, with a frequency of 40kHz, a power of 200W, and an oscillation time of 30 minutes.

7. The method for screening active ingredients in a bone-strengthening preparation based on cell membrane chromatography according to claim 6, characterized in that, The gradient elution method in S2 is as follows: 0-5 min with 100% PBS as the mobile phase; 5-15 min with methanol as the mobile phase for linear gradient elution, wherein the methanol concentration increases from 0% to 30%; 15-20 min with methanol as the mobile phase for linear gradient elution, wherein the methanol concentration increases from 30% to 50%.

8. The method for screening active ingredients in a bone-strengthening preparation based on cell membrane chromatography according to claim 7, characterized in that, The C18 column in S3 has a length of 250 mm, an inner diameter of 4.6 mm, and a packing particle size of 5 μm.

9. The method for screening active ingredients in a bone-strengthening preparation based on cell membrane chromatography according to claim 8, characterized in that, The gradient elution method in S3 is as follows: during the period of 0–10 minutes, the mobile phase B is linearly increased from 5% to 30%; during the period of 10–20 minutes, the mobile phase B is linearly increased from 30% to 60%.

10. The method for screening active ingredients in a bone-strengthening preparation based on cell membrane chromatography according to claim 9, characterized in that, The standard in S4 is icariin, psoralen, pinoresinol diglucoside, and ferulic acid.

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