In-situ kidney perfusion solid-phase chromatography for screening active components of traditional Chinese medicines against diabetic nephropathy and application thereof

Abstract

The application discloses an in-situ kidney perfusion solid-phase chromatography method for screening active components of traditional Chinese medicines against diabetic nephropathy and application thereof. The method comprises the following steps: establishing a fingerprint of a pueraria extract by liquid chromatography-mass spectrometry; establishing a diabetic nephropathy SD rat model and carrying out serum pharmacology research; screening effective components in the pueraria extract by using kidney solid-phase of the SD rat; eluting unbound components; dissociating, preparing kidney homogenate samples and detecting the bound components by liquid chromatography-mass spectrometry. The kidney perfusion solid-phase chromatography technology of the diabetic nephropathy rats and normal rats is used as a kidney perfusion model for initial screening of components against diabetic nephropathy, a new method for screening active components of traditional Chinese medicines with the function of resisting diabetic nephropathy is provided, and the method has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of screening technology for active ingredients of traditional Chinese medicine, and in particular to an in situ renal perfusion solid-phase chromatography method for screening active ingredients of traditional Chinese medicine for treating diabetic nephropathy and its application. Background Technology

[0002] Diabetic nephropathy is the most common microvascular complication of diabetes. Its pathogenesis is complex and not mediated by a single abnormal pathway. The high glucose environment sequentially activates the renal oxidative stress response and inflammatory cascade response, inducing glomerular podocyte damage and renal tubular epithelial cell epithelial-mesenchymal transition, leading to a large amount of abnormal accumulation of renal extracellular matrix. This gradually causes glomerular mesangial expansion, glomerular sclerosis, and renal tubular-interstitial fibrosis. Multiple targets, pathways, and cells are involved in the disease progression, involving a comprehensive disruption of renal targets such as membrane receptors, intracellular signaling proteins, and organelle functional enzymes. This makes the precise screening of the effective components of traditional Chinese medicine for diabetic nephropathy extremely difficult.

[0003] Traditional ex vivo kidney perfusion techniques primarily involve placing an isolated kidney in a perfusion device and using a buffer solution as the perfusion medium to conduct experimental analysis, thereby exploring the interaction between drug components and kidney tissue. While this method can eliminate interference from the body's circulatory system and metabolic processes, it still has significant limitations: the kidney used in the experiment is removed from its original physiological environment, disrupting the complete tissue microcirculation, intercellular signaling, and overall physiological regulatory system, making it impossible to replicate the kidney's true state in vivo. The expression and activation of numerous target proteins and functional proteins in kidney tissue are highly dependent on the body's overall physiological regulation and intercellular synergy. A simple ex vivo perfusion model struggles to maintain the natural activity and function of these targets, easily leading to the ineffective recognition of some specific effector components. Furthermore, during prolonged perfusion, the isolated kidney is susceptible to various factors such as perfusion fluid composition, pH, temperature, perfusion flow rate, and pressure, resulting in a continuous decline in tissue activity and poor experimental stability and reproducibility. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide an in situ renal perfusion solid phase chromatography method for screening active ingredients of traditional Chinese medicine for anti-diabetic nephropathy and its application, in order to solve the above-mentioned technical problems.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a renal solid-phase chromatography method for screening active components of traditional Chinese medicine for treating diabetic nephropathy, comprising the following steps: S1. Fingerprint chromatograms of the extracts of the Chinese herbal medicines to be screened were established by high performance liquid chromatography (HPLC) coupled with mass spectrometry; S2. Establish a diabetic nephropathy SD animal model; S3. Oral renal perfusion was performed on the diabetic nephropathy SD animals and normal animals respectively to screen the effect components of the Chinese medicine extract that bind to the kidney; S4. Elute components that have not bound to the kidneys and discard the eluent; S5. Dissociate to obtain the active ingredient dissociation solution and prepare kidney homogenate samples; S6. Perform HPLC analysis on the obtained active ingredient dissociation solution and kidney homogenate sample to obtain the first analytical result and the second analytical result; S7. By comparing the first analysis result, the second analysis result, and the absorption peaks in the fingerprint spectrum of the traditional Chinese medicine extract, the characteristic peaks of the components that specifically bind to animal kidneys are determined, and the characteristic peaks of the active ingredients are obtained, thereby screening out the active ingredients of traditional Chinese medicine for anti-diabetic nephropathy.

[0006] The renal solid-phase chromatography method for screening active components of traditional Chinese medicine for diabetic nephropathy, wherein the analytical conditions of the HPLC are as follows: The organic phase is acetonitrile; the aqueous phase is 0.1% formic acid solution. Gradient elution is used, and the steps are as follows: The organic phase concentration was 11-12% from 0 to 7 min; 12-13% from 7 to 10 min; 13-14% from 10 to 12 min; 14-15% from 12 to 16 min; 15-18% from 16 to 20 min; 18-32% from 20 to 30 min; 32-35% from 30 to 38 min; 35-45% from 38 to 43 min; 45-55% from 43 to 50 min; 45-55% from 50 to 55 min; and 55-80% from 55 to 60 min. The concentrations of both the organic and aqueous phases are volume percentages. Injection volume: 20 μL; column temperature: 25℃; detection wavelength: 220 nm; flow rate: 1 mL / min; the HPLC column used is a C18 column.

[0007] The renal solid-phase chromatography method for screening active components of traditional Chinese medicine for diabetic nephropathy is described above, wherein the diabetic nephropathy SD animal model is a diabetic nephropathy SD rat model.

[0008] The renal solid-phase chromatography method for screening active components of traditional Chinese medicine for treating diabetic nephropathy includes the following steps in establishing the diabetic nephropathy SD rat model: Male SD rats were fasted for 12 hours and then injected intraperitoneally with streptozotocin (STZ) solution at a concentration of 60 mg / kg for 3 consecutive days. Starting from the third day after the last injection, the rats' body weight was monitored daily, and fasting blood glucose was measured by blood collection from the tail vein. If the fasting blood glucose level was higher than 16.7 mmol / L for three consecutive days, the diabetic nephropathy SD rat model was initially established. Afterward, the rats continued to eat and drink normally. After 4 weeks of feeding, 24-hour urine was collected, urine volume was recorded, and the levels of urinary protein and urinary β-microglobulin were measured. The success of the model was determined based on the urine volume, urinary protein, and urinary β-microglobulin levels.

[0009] The renal solid-phase chromatography method for screening active components of traditional Chinese medicine for treating diabetic nephropathy includes the following steps in the orthotopic renal perfusion method for the diabetic nephropathy SD rat model and normal rats: S31. Diabetic nephropathy SD rat model and normal rats were administered the same dose of Chinese herbal extract and water by gavage, respectively. S32. Anesthetize the rats and cut open their skin to expose the kidneys, abdominal aorta, and vena cava; S33. Tie a knot in the vena cava above the kidney with a thread to prevent blood from the chest cavity from flowing into the renal vein; S34. Following the abdominal aorta of the rat upwards, tie the abdominal aorta segment above the two kidneys with a suture to prevent blood from the chest cavity from flowing into the renal artery, thus avoiding massive bleeding and death of the rat when the blood vessel is cut open below for cannulation later. S35. Ligate and tie the mesenteric artery near the kidneys in the small and large intestines with a suture to prevent the perfusion fluid from flowing into the mesenteric artery and entering the small and large intestines; S36. Make a small incision in the abdominal aorta below the rat's kidney, insert a PEG medical silicone tube into the abdominal aorta, suture and ligate it, and perform the same operation on the vena cava. S37. After the cannulation and ligation are completed, immediately turn on the irrigation equipment to start irrigation, which will continue for 25 minutes.

[0010] The renal solid-phase chromatography method for screening active components of traditional Chinese medicine for anti-diabetic nephropathy, wherein in step S4, the elution is performed by rinsing with perfusion fluid for at least 25 minutes.

[0011] The renal solid-phase chromatography method for screening active components of traditional Chinese medicine for anti-diabetic nephropathy, wherein the perfusion fluid is KH perfusion buffer.

[0012] The renal solid-phase chromatography method for screening active ingredients of traditional Chinese medicine for diabetic nephropathy, wherein in step S5, a citric acid buffer solution is used for perfusion for 25-30 minutes, and the dissociation solution of the active ingredient is collected.

[0013] The renal solid-phase chromatography method for screening active components of traditional Chinese medicine for anti-diabetic nephropathy, wherein in S1, the preparation method of the extract of the traditional Chinese medicine to be screened is as follows: take the traditional Chinese medicine to be screened, add six times the amount of ethanol and water, weigh it, heat it under reflux, cool it to room temperature, weigh it again, replenish the lost weight with ethanol and water, filter it through a 0.22μm microporous membrane, and obtain the ethanol extract of the traditional Chinese medicine to be screened.

[0014] The second aspect of this invention provides the application of the renal solid-phase chromatography method for screening active components of traditional Chinese medicine for anti-diabetic nephropathy in the screening of renal active components of traditional Chinese medicine.

[0015] Beneficial Effects: The renal solid-phase chromatography method for screening active components of traditional Chinese medicine (TCM) in the treatment of diabetic nephropathy provided by this invention employs in situ renal perfusion technology. Furthermore, the in situ perfusion model can more realistically simulate the in vivo physiological environment, possessing significant advantages for in situ research. The in situ renal perfusion solid-phase chromatography technology used in this invention is an optimized upgrade of traditional ex vivo renal perfusion, combining the in situ renal perfusion system with chromatographic detection technology. This allows for the full binding of TCM active components to in situ targets in the kidney while maximally replicating the in vivo physiological environment of the kidney. The screening process first removes components that have not specifically bound through in situ perfusion, and then dissociates and detects the active components bound to the kidney tissue. This technology maximally preserves the overall physiological structure of the kidney and the activity of pathologically related biological targets, completely replicating the in vivo pathological microenvironment of kidney disease. This represents a major innovation and improvement over traditional ex vivo renal perfusion chromatography technology.

[0016] Furthermore, this invention utilizes the kidneys of diabetic nephropathy rats for screening, enabling preliminary screening of substances with potential renal activity. This represents a breakthrough in the screening of renal active ingredients and has promising application prospects. Verification using kudzu root extract effectively demonstrates that the technical solution of this invention can effectively screen for active ingredients in traditional Chinese medicine for treating diabetic nephropathy. Attached Figure Description

[0017] Figure 1 The images show the HPLC chromatograms of kudzu root extract. In the figures, A is the HPLC chromatogram of the standard; B is the HPLC chromatogram of the kudzu root extract.

[0018] Figure 2 The images show the total ion chromatograms of kudzu root extract. A represents the positive ion mode ESI mass spectrum of the kudzu root extract; B represents the negative ion mode ESI mass spectrum of the kudzu root extract.

[0019] Figure 3 The biochemical status indicators of rats in the DKD group and the normal group are as follows: A represents body weight change; B represents blood glucose change; C represents total 24-hour urinary protein; D represents 24-hour urinary β-microglobulin content; and E represents the kidney index.

[0020] Figure 4The images show ESI mass spectra of the dissociation fluid from kudzu root rats in negative ion mode. Specifically, A represents the negative ion mass spectrum of the kudzu root extract; B represents the negative ion mass spectrum of the dissociation fluid from normal kudzu root rats; C represents the negative ion mass spectrum of the dissociation fluid from kudzu root DKD rats; D represents the negative ion spectrum of the last perfusion fluid from normal kudzu root rats; and E represents the negative ion spectrum of the last perfusion fluid from kudzu root DKD rats.

[0021] Figure 5 The images show positive ion mode ESI mass spectra of the kudzu root rat dissociation fluid. Specifically, A represents the positive ion mass spectrum of the kudzu root extract; B represents the positive ion mass spectrum of the kudzu root dissociation fluid from normal rats; C represents the positive ion mass spectrum of the kudzu root DKD rat dissociation fluid; D represents the positive ion spectrum of the last perfusion fluid from normal rats; and E represents the positive ion spectrum of the last perfusion fluid from kudzu root DKD rats.

[0022] Figure 6 The images show ESI mass spectra of kudzu root rat kidney homogenates in negative ion mode. Specifically, A represents the negative ion mass spectrum of kudzu root extract; B represents the negative ion mass spectrum of normal kudzu root rat kidney homogenate; C represents the negative ion mass spectrum of kudzu root DKD rat kidney homogenate; D represents the negative ion spectrum of the last dissociation solution from normal kudzu root rats; and E represents the negative ion spectrum of the last dissociation solution from DKD rats.

[0023] Figure 7 ESI mass spectra of kudzu root rat kidney homogenate in positive ion mode. A represents the positive ion mass spectrum of kudzu root extract; B represents the positive ion mass spectrum of kudzu root normal rat kidney homogenate; C represents the positive ion mass spectrum of kudzu root DKD rat kidney homogenate; D represents the positive ion spectrum of the last dissociation solution from kudzu root normal rats; and E represents the positive ion spectrum of the last dissociation solution from kudzu root DKD rats.

[0024] Figures 4 to 7 In the middle, peak 14 is puerarin; M1-M72 are metabolic components.

[0025] Figure 8 This study investigated the repair effects of renal solidification components on acetone-induced renal tubular epithelial cells. A represents the damaging effects of different concentrations of acetone on renal tubular epithelial cells; B represents puerarin; and C represents total flavonoids from puerarin. Compared with the control group, ### represents p<0.001; compared with the model group, This means P < 0.05. This means P < 0.01. This means P < 0.001.

[0026] Figure 9 This study investigated the inhibitory effect of renal solidification components on an excessively proliferating glomerular mesangial cell model. A represents puerarin; B represents total puerarin flavonoids. Compared with the control group, ### represents p<0.001; compared with the model group, This means P < 0.05. This means P < 0.01. This means p < 0.001. Detailed Implementation

[0027] This invention provides an in situ renal perfusion solid-phase chromatography method for screening active components of traditional Chinese medicine for diabetic nephropathy and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0028] The following uses kudzu root as the Chinese herbal medicine to be screened to illustrate the technical solution of this invention.

[0029] The kudzu root (Pueraria lobata) used was purchased from Anguo Juyitang Pharmaceutical Co., Ltd. (item number: 20230004).

[0030] Example 1: Establishment of an in-situ renal perfusion solid-phase chromatography model for screening renal active components of traditional Chinese medicine 1. Establishment of kudzu root fingerprint spectrum Sample preparation: Take 4.0g of kudzu root, accurately weigh it, place it in a 150 mL stoppered conical flask, accurately add 6 times the amount of 50% ethanol water, seal it, weigh it, heat it under reflux for 45 min, cool it to room temperature, weigh it again, make up the lost weight with 50% ethanol water, and filter it through a 0.22μm microporous membrane.

[0031] Preparation of standard: Accurately weigh 0.3 mg of puerarin and dissolve it in 1 mL of methanol to obtain a 0.3 mg / mL standard. Filter the standard through a 0.22 μL microporous membrane before loading.

[0032] Liquid chromatography conditions: organic phase: acetonitrile; aqueous phase: 0.1% formic acid solution; gradient elution: 0–7 min (organic phase: 11–12%), 7–10 min (organic phase: 12–13%), 10–12 min (organic phase: 13–14%), 12–16 min (organic phase: 14–15%), 16–20 min (organic phase: 15–18%), 20–30 min (organic phase: 18–32%), 30 min… ~38 min (organic phase: 32-35%), 38-43 min (organic phase: 35-45%), 43-50 min (organic phase: 45-55%), 50-55 min (organic phase: 45-55%), 55-60 min (organic phase: 55-80%), all of which are volume percentages; injection volume: 10 μL; column temperature: 25-35℃; detection wavelength: 250 nm; flow rate: 1 mL / min.

[0033] Liquid chromatography-mass spectrometry (LC-MS): Thermo Fisher Scientific LTQ-Obitrap mass spectrometer; Column: Platisil C18 (5μm 250 x 4.6mm).

[0034] Mass spectrometry conditions: spray voltage 2.5 kV, sheath gas (N2) pressure 75 arb, auxiliary gas (N2) pressure 20 arb, make-up gas flow rate 4 arb, capillary temperature 400℃. All sample data were acquired using full scan mode at a resolution of 70000 FWHM. The first-stage full scan mass number range was m / z 80-1200; the second stage used data-dependent scanning, setting precursor ions with a threshold of 1000 or higher as the scan targets, dynamic exclusion time set to 15 s, number of repeated scans set to 2, repeated scan time set to 15 s, and collision-induced dissociation voltage set to 35 V.

[0035] Kudzu fingerprint spectrum as follows Figure 1 , Figure 2 As shown, where, Figure 1 A is the HPLC chromatogram of the standard; Figure 1 B is the HPLC chromatogram of kudzu root extract; Figure 2 A is the positive ion mode ESI mass spectrum of kudzu root extract; Figure 2 B is the ESI mass spectrum of kudzu root extract in negative ion mode.

[0036] 2. Establishment of a rat model of diabetic nephropathy Eight-year-old male SD rats were randomly divided into a control group and a model group. The control group was fed a normal diet, while the model group was fasted for 12 hours (with no restriction on water) and then injected intraperitoneally with streptozotocin (STZ) solution (60 mg / kg) for 3 consecutive days. The control group was simultaneously injected with an equal volume of sodium citrate buffer. Starting from the 3rd day after the last injection, the rats' body weight was monitored daily, and fasting blood glucose was measured by collecting blood from the tail vein. If the fasting blood glucose level was higher than 16.7 mmol / L for 3 consecutive days, the diabetic model was considered to have been initially established successfully. Thereafter, the rats continued to eat and drink normally. After 4 weeks of feeding, they were transferred to metabolic cages to collect 24-hour urine. The urine volume was recorded, and the levels of urinary protein and urinary β-microglobulin were measured to further evaluate whether the diabetic nephropathy model had been successfully established.

[0037] like Figure 3 As shown, the model group rats exhibited symptoms such as polydipsia, polyphagia, and increased urine output. Blood glucose levels remained consistently above 16.7 mmol / L. The levels of urinary protein and β-microglobulin, indicators of kidney damage, were significantly increased, and the kidney index was also much higher than that of the normal group rats. All of these indicators are consistent with the clinical manifestations of DKD. Therefore, it can be confirmed that the rat DKD model has been established and can be used for the following experiments.

[0038] 3. Solid-phase chromatography experiment of in situ renal perfusion in diabetic nephropathy rats (1) Preparation of kudzu root extract: Take 4.0g of kudzu root, accurately weigh it, place it in a 150 mL stoppered conical flask, accurately add 6 times 50% ethanol water, seal it, weigh it, heat it under reflux for 45 min, cool it to room temperature, weigh it again, make up the lost weight with 50% ethanol water, and filter it through a 0.22 μm microporous membrane.

[0039] (2) Gavage administration: Six normal rats were randomly selected and assigned to the kudzu root group and the normal blank control group, with three rats in each group. Similarly, six rats with successful DKD modeling were randomly selected and assigned to the DKD kudzu root group and the DKD blank control group, with three rats in each group. All rats were fasted for 12 hours before the experiment, but water was allowed. The kudzu root group was administered kudzu root extract (30 g / kg / time based on the crude drug amount) by gavage, while the blank control group was administered an equal volume of water by gavage.

[0040] (3) Perfusion: SD rats (normal or DKD) were administered the same dose of kudzu root aqueous extract and water by gavage. One hour later, 4 mL / kg of 10% chloral hydrate solution was injected intraperitoneally. After anesthesia, the rats were fixed supine on the operating table. The abdominal hair was moistened with povidone-iodine. The skin was cut near the kidneys below the thoracic cavity with surgical scissors. The small and large intestines were gently separated, and the mucosa was separated to expose the kidneys, abdominal aorta, and vena cava. A surgical suture was gently passed through the mucosa below the vena cava connected above the kidneys with hemostatic forceps. The suture was tied at this point in the vena cava to prevent blood from the thoracic cavity from flowing into the renal vein. The abdominal aorta was ligated and tied upwards along the abdominal aorta above the two kidneys to prevent blood from the thoracic cavity from flowing into the renal artery (to avoid massive bleeding and rat death when cutting open the blood vessels below for cannulation). The mesenteric arteries near the small and large intestines near the kidneys were also ligated and tied (to prevent the perfusion fluid from flowing into the mesenteric arteries and into the small and large intestines, disrupting their physiological environment). Next, carefully separate the vena cava and abdominal aorta below the kidney using microsurgical hemostatic forceps. Gently lift the abdominal aorta with forceps, and make a small incision in the aorta with microsurgical scissors. Use sterile cotton to absorb any blood that flows out to avoid obstructing the view of the catheter. Insert the PEG medical silicone tube into the abdominal aorta and immediately ligate it at this point with surgical sutures to secure the tube and ensure that the perfusion fluid does not spill out. Repeat the above procedure for the vena cava. After the catheter is inserted and ligated, immediately turn on the perfusion device to begin perfusion, which will continue for 25 minutes.

[0041] (4) Dissociation and preparation of kidney homogenate: After eluting the unbound components in step (3), the solution was replaced with citric acid buffer solution and perfused for 25 minutes. The dissociation solution was collected, the kidney was removed and ground with physiological saline. The dissociation solution and kidney homogenate are the active ingredients.

[0042] (5) HPLC analysis of the active ingredient dissociation solution: The active ingredient dissociation solution and kidney homogenate were evaporated to dryness in a water bath and then reconstituted to 200 μL. HPLC analysis was performed. By comparing the absorption peaks in the fingerprint spectra of the active ingredient dissociation solution, the last eluent in step (3), and the kudzu root extract obtained in step (1), the characteristic peaks with adsorption to the kidney were determined, and the characteristic peaks of the active ingredient were obtained.

[0043] The results are as follows Figure 2 As shown, 57 peaks were obtained from the kudzu root extract, and 50 components were identified by mass spectrometry (see Table 1). All identified compounds were reported in the component analysis of kudzu root. To verify the accuracy, puerarin (peak 14), a representative component of kudzu root, was used as a standard control (see Table 1). Figure 1 The results of the mass spectrometry analysis were consistent with those of the standard sample, indicating that the mass spectrometry results are reliable and accurate.

[0044] like Figure 4As shown, no peaks were observed in the final perfusion chromatogram, indicating that the non-adsorbed components were completely washed away after 25 minutes of perfusion. Comparison of the kudzu root extract with the active ingredient dissociation solution and kidney homogenate confirmed that peak 14 was puerarin, and most of the remaining characteristic peaks were flavonoids. These characteristic peaks represent the group of effector components that bind to the rat kidney target site. The blank control experiment showed no peaks in the dissociation solution and kidney homogenate chromatograms, ruling out interference from endogenous substances and further demonstrating that the substances in the active ingredient dissociation solution are indeed potential renal active components in the herb that have an affinity for and adsorption to the renal membrane.

[0045] Modern research has confirmed that flavonoids such as puerarin in kudzu root have a clear protective effect against diabetic nephropathy, which is consistent with the conclusions of this invention.

[0046] Table 1

[0047]

[0048]

[0049]

[0050] Example 2: In vitro efficacy evaluation of solid-phase components of kudzu root in situ renal perfusion Weigh kudzu root, add 70% ethanol at a material-to-liquid ratio of 1:40 (g:mL), and extract by reflux at 90℃ for 120 min. Filter, concentrate under reduced pressure, and dry. Accurately weigh the total flavonoid extract of kudzu root, dissolve, and dilute to a final volume to prepare a 0.5 mg / mL solution for later use.

[0051] Accurately weigh an appropriate amount of puerarin reference standard, place it in a volumetric flask, add an appropriate amount of methanol to dissolve it, dilute to the mark, and shake well to prepare a stock solution of puerarin reference standard at a certain concentration. Accurately pipette an appropriate amount of the stock solution and dilute it stepwise with the appropriate solvent to obtain a series of puerarin reference standard solutions of different concentrations (0.2, 0.3, 0.4, 0.5, 0.6 mg / mL) for later use.

[0052] Using an ELISA reader with methanol as a blank control, the absorbance of puerarin standard solutions at various concentrations was measured at a selected wavelength of 250 nm. A linear regression analysis was performed with puerarin concentration (C) on the x-axis and absorbance (A) on the y-axis to plot a standard curve and obtain the regression equation. A suitable amount of the test solution was precisely pipetted and subjected to color development or direct measurement using the same method as for plotting the standard curve, and the absorbance was recorded. The total flavonoid concentration equivalent to puerarin in the test solution was calculated by substituting the absorbance into the regression equation.

[0053] A linear regression was performed with the concentration of puerarin standard as the x-axis and absorbance as the y-axis, yielding the standard curve regression equation: A = 3.0321C + 0.3717, and the correlation coefficient R0. 2 =0.9998. The results show that puerarin exhibits a good linear relationship between concentration and absorbance in the range of 0.2–0.6 mg / mL, and can be used for the quantitative determination of total flavonoid content.

[0054] The absorbance of the total flavonoid solution from kudzu root was substituted into the standard curve regression equation for calculation. The results showed that the total flavonoid concentration in the test solution was 0.341 mg / mL (calculated as puerarin), corresponding to a total flavonoid content of 68.27% in the extract. Calculations showed that 200 μL of 625 μM puerarin corresponds to a total flavonoid extract of 761.7 μg / mL. Subsequent in vitro cell experiments selected 761.7 μg / mL as the maximum concentration of total flavonoids for cellular efficacy validation.

[0055] Human renal tubular epithelial cells (HK2) were processed at a ratio of 1×10⁻⁶. 5 Cells were seeded at a density of cells / well in 96-well plates, with three replicates per group. When cells reached 80% confluence, the drug was administered. The experimental groups were as follows: Control group, Model group (MG 0.64 mmol / L), and 0.64 mmol / L + various concentrations of immobilized monomeric compounds. After 24 hours of treatment, cell viability was assessed using a CCK-8 assay kit. Cell viability (%) = (OD value of experimental group - OD value of blank well) / (OD value of control group - OD value of blank well) × 100%.

[0056] like Figure 8 As shown in Figure A, concentration gradients ranging from 0.04 to 2.56 mM all induced varying degrees of damage to renal tubular epithelial cells. A 0.64 mM acetone aldehyde was selected as the concentration for inducing a renal tubular epithelial cell injury model.

[0057] from Figure 8 B and Figure 8 Among the two representative components observed in C, puerarin (0.2 μM) and total flavonoids from puerarin (0.0448 μg / mL) showed significant inhibitory effects.

[0058] After human glomerular mesangial cells (HRMCs) grew well and were synchronized for 24 hours, cell seeding began. 8000 cells were seeded per well in 96-well plates. After reaching a cell density of 60-70% per well, cell drug administration began. Three groups were established: a low-glucose group (5.5 mM glucose); a high-glucose group (25 mM glucose); and groups treated with 25 mmol / L glucose plus various concentration gradients of renal perfusion solid-phase monomers. After 24 hours of incubation, cell growth was measured using a CCK-8 assay kit to observe the therapeutic effect of the renal perfusion solid-phase monomer components on high-glucose-induced excessive proliferation of glomerular mesangial cells.

[0059] Compared with the normal group, high concentrations of 25 mM glucose significantly promoted excessive proliferation of glomerular mesangial cells. Figure 9 As shown, puerarin at concentrations of 0.04–625 μM and total puerarin flavonoids at concentrations of 6.0936–761.7000 μg / mL significantly inhibited high glucose-induced excessive proliferation of glomerular mesangial cells. Among these representative compounds, puerarin showed the best effect.

[0060] Example 2 used two cell models of diabetic nephropathy to perform pharmacodynamic assays on puerarin and total flavonoids from kudzu root, which were specifically bound by in situ renal perfusion solid-phase chromatography, to verify their activity. The results showed that puerarin and total flavonoids from kudzu root have the potential for practical application in the treatment of diabetic nephropathy, which is consistent with current research confirming that puerarin and other flavonoids in kudzu root have a clear protective effect against diabetic nephropathy, indicating that the method of the present invention is reliable.

[0061] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solution and inventive concept of the present invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A renal solid-phase chromatography method for screening active components of traditional Chinese medicine for treating diabetic nephropathy, characterized in that, Includes the following steps: S1. Fingerprint chromatograms of the extracts of the Chinese herbal medicines to be screened were established by high performance liquid chromatography (HPLC) coupled with mass spectrometry; S2. Establish a diabetic nephropathy SD animal model; S3. Oral renal perfusion was performed on the diabetic nephropathy SD animals and normal animals respectively to screen the effect components of the Chinese medicine extract that bind to the kidney; S4. Elute components that have not bound to the kidneys and discard the eluent; S5. Dissociate to obtain the active ingredient dissociation solution and prepare kidney homogenate samples; S6. Perform HPLC analysis on the obtained active ingredient dissociation solution and kidney homogenate sample to obtain the first analytical result and the second analytical result; S7. By comparing the first analysis result, the second analysis result, and the absorption peaks in the fingerprint spectrum of the traditional Chinese medicine extract, the characteristic peaks of the components that specifically bind to animal kidneys are determined, and the characteristic peaks of the active ingredients are obtained, thereby screening out the active ingredients of traditional Chinese medicine for anti-diabetic nephropathy.

2. The renal solid-phase chromatography method for screening active components of traditional Chinese medicine for anti-diabetic nephropathy according to claim 1, characterized in that, The analytical conditions for the HPLC are as follows: The organic phase is acetonitrile; the aqueous phase is 0.1% formic acid solution. Gradient elution is used, and the steps are as follows: The organic phase concentration was 11-12% from 0 to 7 min; 12-13% from 7 to 10 min; 13-14% from 10 to 12 min; 14-15% from 12 to 16 min; 15-18% from 16 to 20 min; 18-32% from 20 to 30 min; 32-35% from 30 to 38 min; 35-45% from 38 to 43 min; 45-55% from 43 to 50 min; 45-55% from 50 to 55 min; and 55-80% from 55 to 60 min. The concentrations of both the organic and aqueous phases are volume percentages. Injection volume: 20 μL; Column temperature: 25℃; detection wavelength: 220nm; flow rate: 1mL / min; the HPLC column used is a C18 column.

3. The renal solid-phase chromatography method for screening active components of traditional Chinese medicine for anti-diabetic nephropathy according to claim 1, characterized in that, The diabetic nephropathy SD animal model is a diabetic nephropathy SD rat model.

4. The renal solid-phase chromatography method for screening active components of traditional Chinese medicine for anti-diabetic nephropathy according to claim 3, characterized in that, The method for establishing the diabetic nephropathy SD rat model includes the following steps: Male SD rats were fasted for 12 hours and then injected intraperitoneally with streptozotocin (STZ) solution at a concentration of 60 mg / kg for 3 consecutive days. Starting from the third day after the last injection, the rats' body weight was monitored daily, and fasting blood glucose was measured by blood collection from the tail vein. If the fasting blood glucose level was higher than 16.7 mmol / L for three consecutive days, the diabetic nephropathy SD rat model was initially established. Afterward, the rats continued to eat and drink normally. After 4 weeks of feeding, 24-hour urine was collected, urine volume was recorded, and the levels of urinary protein and urinary β-microglobulin were measured. The success of the model was determined based on the urine volume, urinary protein, and urinary β-microglobulin levels.

5. The renal solid-phase chromatography method for screening active components of traditional Chinese medicine for anti-diabetic nephropathy according to claim 3, characterized in that, The orthotopic renal perfusion method for the diabetic nephropathy SD rat model and normal rats includes the following steps: S31. Diabetic nephropathy SD rat model and normal rats were administered the same dose of Chinese herbal extract and water by gavage, respectively. S32. Anesthetize the rats and cut open their skin to expose the kidneys, abdominal aorta, and vena cava; S33. Tie a knot in the vena cava above the kidney with a thread to prevent blood from the chest cavity from flowing into the renal vein; S34. Following the abdominal aorta of the rat upwards, tie the abdominal aorta segment above the two kidneys with a suture to prevent blood above the chest cavity from flowing into the renal artery, thus avoiding massive bleeding and death of the rat when the blood vessel is cut open and cannulated later. S35. Ligate and tie the mesenteric artery near the kidneys in the small and large intestines with a suture to prevent the perfusion fluid from flowing into the mesenteric artery and entering the small and large intestines; S36. Make a small incision in the abdominal aorta below the rat's kidney, insert a PEG medical silicone tube into the abdominal aorta, suture and ligate it, and perform the same operation on the vena cava. S37. After the cannulation and ligation are completed, immediately turn on the irrigation equipment to start irrigation, which will continue for 25 minutes.

6. The renal solid-phase chromatography method for screening active components of traditional Chinese medicine for anti-diabetic nephropathy according to claim 1, characterized in that, In step S4, the elution process involves rinsing with perfusion fluid for at least 25 minutes.

7. The renal solid-phase chromatography method for screening active components of traditional Chinese medicine for anti-diabetic nephropathy according to claim 6, characterized in that, The perfusion fluid is KH perfusion buffer.

8. The renal solid-phase chromatography method for screening active components of traditional Chinese medicine for anti-diabetic nephropathy according to claim 1, characterized in that, In step S5, a citric acid buffer solution is used for perfusion for 25–30 minutes to collect the dissociation solution of the active ingredient.

9. The renal solid-phase chromatography method for screening active components of traditional Chinese medicine for anti-diabetic nephropathy according to claim 1, characterized in that, In S1, the preparation method of the herbal extract to be screened is as follows: Take the herbal extract to be screened, add six times the amount of ethanol and water, weigh it, heat it under reflux, cool it to room temperature, weigh it again, replenish the lost weight with ethanol and water, filter it through a 0.22μm microporous membrane, and obtain the ethanol extract of the herbal extract to be screened.

10. The application of the renal solid-phase chromatography method for screening active components of traditional Chinese medicine for anti-diabetic nephropathy as described in any one of claims 1-9 in the screening of renal active components of traditional Chinese medicine.

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