Method for detecting effective components of kidney-tonifying, mass-eliminating and wind-dispelling prescription for treating diabetic nephropathy

Abstract

The invention belongs to the technical field of analysis of traditional Chinese medicine preparations, and discloses a method for detecting effective components of a kidney-tonifying, mass-eliminating and wind-dispelling prescription for treating diabetic nephropathy. According to the method, an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF / MS) and an ultra-high performance biological inert liquid chromatography-triple quadrupole mass spectrometry (ACQUITY Premier / Xevo TQ-XS) are adopted to carry out system identification and quantitative analysis on chemical components in the formula for tonifying kidney, eliminating diseases and dispelling wind. By establishing a characteristic fingerprint spectrum and a multi-reaction monitoring (MRM) mode, accurate qualitative and quantitative detection of main active ingredients is realized, and the pharmacodynamic material basis of the kidney-tonifying, mass-eliminating and wind-dispelling prescription for treating diabetic nephropathy is defined. According to the method, a comprehensive, scientific and repeatable detection basis is provided for internal quality control of the kidney-tonifying, mass-eliminating and wind-dispelling prescription, and the safety and effectiveness of clinical application of the traditional Chinese medicine compound are guaranteed.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation analysis technology, and discloses a method for detecting the effective components of Yishen Xiaozheng Qufeng Formula for the treatment of diabetic nephropathy. Background Technology

[0002] Diabetic nephropathy is one of the most common and harmful chronic complications of diabetes. Its pathogenesis involves multiple pathological processes, including inflammatory response, oxidative stress, glomerular sclerosis, and energy metabolism disorders. Current Western medicine treatments mainly rely on drugs that lower blood sugar, blood pressure, and protein levels (such as SGLT2 inhibitors, ACEIs / ARBs). While these can slow disease progression, they cannot fundamentally reverse kidney damage.

[0003] The Kidney-Nourishing, Mass-Dispelling, and Wind-Expelling Formula is an in-house traditional Chinese medicine compound developed by Hangzhou Municipal Hospital of Traditional Chinese Medicine. It originates from the "Invigorating Qi and Blood, Dispelling Wind and Unblocking Collaterals" academic system of Professor Wang Yongjun of the Zhejiang School of Traditional Chinese Medicine. The formula primarily consists of Astragalus membranaceus, Panax notoginseng, Euryale ferox, Lonicera japonica, Spatholobus suberectus, Sparganium stoloniferum, and Dioscorea opposita, possessing the functions of invigorating Qi and nourishing Yin, dispelling wind and promoting blood circulation, and unblocking collaterals and eliminating masses. Clinically, it is used for patients with diabetic nephropathy, significantly improving proteinuria, glomerular filtration rate, and renal function indicators.

[0004] However, the current quality control of this compound still mainly relies on traditional Chinese medicine identification and single-component detection methods, which cannot accurately reflect the chemical composition and efficacy characteristics of the compound.

[0005] Existing detection methods, such as high-performance liquid chromatography or ultraviolet spectrophotometry, can only determine some indicative components, and have the following limitations: 1. Limited detection range, making it difficult to fully reveal the complex chemical components of compound preparations; 2. Insufficient resolution and sensitivity, prone to multi-component co-elution; 3. Lack of a systematic quantitative framework makes it impossible to establish a correlation between ingredients and therapeutic effects; 4. Lack of standardized quality control methods affects batch-to-batch consistency and safety of formulations.

[0006] Therefore, there is an urgent need to establish a highly sensitive, high-resolution, and repeatable detection method that can comprehensively identify the main active ingredients in Yishen Xiaozheng Qufeng Formula, clarify its material basis for treating diabetic nephropathy, and provide a scientific basis for the modern quality control and pharmacological mechanism research of traditional Chinese medicine compound formulas. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention discloses a method for detecting the effective components of Yishen Xiaozheng Qufeng Formula for the treatment of diabetic nephropathy. The method uses UPLC-Q-TOF / MS to identify the effective active components in Yishen Xiaozheng Qufeng Formula, and further identifies the active components using UPLC-TQ / MS, achieving a comprehensive evaluation of the effective components of Yishen Xiaozheng Qufeng Formula and ensuring the safety and efficacy of this traditional Chinese medicine compound in clinical use.

[0008] The specific technical solution is to provide a method for detecting the effective components of Yishen Xiaozheng Qufeng Formula: UPLC-Q-TOF / MS is used to identify the effective active components in the formula, and UPLC-TQ / MS is used to further qualitatively analyze and confirm the above-mentioned monomeric small molecules.

[0009] Furthermore, the method for preparing the sample required for UPLC-Q-TOF / MS of this invention is as follows: (1) Take the powder of Yishen Xiaozheng Qufeng Fang, add 50% methanol, mix well, let stand for 2 minutes, vortex for 30 seconds, let stand for 30 seconds, repeat 5-10 times. (2) Place the mixture in an ice bath and sonicate for 20-30 min, then settle at -20℃ for 30 min; centrifuge at 15000 rpm for 10 min in a pre-cooled centrifuge at 4℃ and collect the supernatant. (3) Transfer the supernatant to a new tube, extract the residue again according to the above steps, and combine the supernatants, with a volume of about 1 mL; (4) If the solution is viscous, add anhydrous ethanol to the supernatant to a final concentration of 80% (v / v), let it stand at -20℃ for 1 h, and then centrifuge at 4℃ and 15000 rpm for 10 min to collect the supernatant. (5) Filter with a 0.22 μm filter membrane and use the filtrate as the sample for the instrument.

[0010] The steps for determining the candidate active ingredients of Yishen Xiaozheng Qufeng Formula by UPLC-Q-TOF / MS include: (1) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the packing material; 0.1% formic acid water and acetonitrile were used as the mobile phase; evaporative light scattering detector was used for detection; only samples could be injected after the suitability was determined to be qualified. (2) Preparation of reference solution: Prepare a 50% (v / v) methanol solution as the reference solution; (3) Preparation of test solution: Take the powder of Yishen Xiaozheng Qufeng Fang, put it in a 10 mL test tube, vortex for 5 min, then sonicate for 30 min, then centrifuge to take the supernatant, filter it through a 0.22 μm filter membrane and bottle it to obtain the test sample; (4) Determination of active ingredients: First, run the reference solution to confirm the baseline. Then, accurately pipette the reference solution and the test solution and inject them into the liquid chromatograph. The injection volume is 2L, the column temperature is 35℃, the sample chamber temperature is 10℃, and the mass accuracy is 0.2ppm. Then run the program to determine the active ingredients.

[0011] The method for detecting the effective components of a kidney-tonifying, phlegm-reducing, and wind-dispelling formula provided by this invention has the following beneficial effects: This invention employs ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF / MS) and ultra-high performance bio-inert liquid chromatography-triple quadrupole mass spectrometry (ACQUITY Premier / Xevo TQ-XS) to systematically identify and quantitatively analyze the chemical components of Yishen Xiaozheng Qufeng Formula, belonging to the field of quality control and pharmacodynamic material basis research of traditional Chinese medicine compound formulas. Using Yishen Xiaozheng Qufeng Formula as the research object, a concentrated extract was obtained through an ethanol / water system. The chemical components of the compound formula were systematically qualitatively analyzed by UPLC-Q-TOF / MS, establishing chemical component spectra and characteristic fingerprints. Subsequently, based on the UPLC-TQ-MS / MS platform, the main active ingredients were quantitatively detected and validated in multiple reaction monitoring (MRM) mode. Through the complementary application of the two mass spectrometry techniques, a systematic study of the compound formula components was achieved, from comprehensive qualitative analysis to precise quantification and methodological validation.

[0012] Method validation results showed that the precision RSD was <2.5%, the repeatability RSD was <3.0%, the spiked recovery rate was 98.5%–102.3%, the linear correlation coefficient R² > 0.995, and the limit of detection was 0.3–0.5 ng / mL. These results indicate that the method has good linearity, precision, stability, and sensitivity, and can achieve accurate quantification and repeatable detection of complex components in traditional Chinese medicine compound prescriptions.

[0013] This invention is the first to apply a dual-platform approach of UPLC-Q-TOF / MS and UPLC-TQ / MS to the systematic quality control of Yishen Xiaozheng Qufeng Formula, forming an integrated methodology system of "high-resolution qualitative analysis + high-sensitivity quantitative analysis + fingerprint spectral evaluation". Methodological validation shows that this system is significantly superior to traditional HPLC in terms of sensitivity, resolution, and repeatability. The results can be directly applied to the establishment of in-hospital formulation quality standards, consistency verification of production processes, and quality control of multi-center clinical research data, demonstrating innovation, practicality, and industrialization potential. Attached Figure Description

[0014] Figure 1 Flowchart of the detection method.

[0015] Figure 2 High-performance liquid chromatogram of the aqueous extract of the kidney-tonifying, mass-dissolving, and wind-dispelling formula.

[0016] Figure 3 Blank solvent high-performance liquid chromatogram.

[0017] Figure 4 High-performance liquid chromatogram of active small molecule monomers in mixed standard solution - 1.

[0018] Figure 5 High-performance liquid chromatogram of active small molecule monomers in mixed standard solution -2.

[0019] Figure 6 Small molecule MS-UPLC-TQ / MS mass spectrometry analysis. Detailed Implementation

[0020] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0021] The flowchart of the detection method for the effective components of the kidney-tonifying, phlegm-reducing, and wind-dispelling formula of this invention is as follows: Figure 1 As shown.

[0022] This invention relates to a kidney-tonifying, mass-eliminating, and wind-dispelling formula comprising the following ingredients by weight: Astragalus membranaceus 30g, Panax notoginseng 3g, Euryale ferox 30g, Lonicera japonica vine 15g, Spatholobus suberectus 15g, Sparganium stoloniferum 15g, and Dioscorea opposita 15g. The above-mentioned medicinal materials are decocted twice with 6 times the amount of water, each time for 60 minutes. The decoctions are combined to obtain a soup, which is then concentrated and dried to obtain the kidney-tonifying, mass-eliminating, and wind-dispelling formula.

[0023] Example 1: Determination of active ingredients in the Kidney-Nourishing, Mass-Eliminating, and Wind-Dispelling Formula Medicines and reagents: Yishen Xiaozheng Qufeng powder, produced by the Department of Pharmacy, Zhejiang University of Traditional Chinese Medicine.

[0024] Experimental apparatus: Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF / MS).

[0025] The sample preparation required for UPLC-Q-TOF / MS is as follows: (1) Take the powder of Yishen Xiaozheng Qufeng Fang, add 50% methanol, mix well, let stand for 2 minutes, vortex for 30 seconds, let stand for 30 seconds, repeat 5-10 times. (2) Place the mixture in an ice bath and sonicate for 20-30 min, then settle at -20℃ for 30 min; centrifuge at 15000 rpm for 10 min in a pre-cooled centrifuge at 4℃ and collect the supernatant. (3) Transfer the supernatant to a new tube, extract the residue again according to the above steps, and combine the supernatants, with a volume of about 1 mL; (4) If the solution is viscous, add anhydrous ethanol to the supernatant to a final concentration of 80% (v / v), let it stand at -20℃ for 1 h, and then centrifuge at 4℃ and 15000 rpm for 10 min to collect the supernatant. (5) Filter with a 0.22 μm filter membrane and use the filtrate as the sample for the instrument.

[0026] 1) Chromatographic conditions and system suitability test 1.1 Liquid Chromatography Conditions Octadecylsilane-bonded silica gel was used as the filler; formic acid-acetonitrile (continuous gradient) was used as the mobile phase; Flow rate: 0.3 mL / min; Column temperature: 35℃; Injection volume: 2 μL; Mobile phase: 0.1% formic acid solution (A) and pure acetonitrile (B). The gradient elution method for 35 minutes is as follows: 0-2 min, 5% acetonitrile (B); 2-32 min, 5-100% acetonitrile (B); 32-33 min, 100% acetonitrile (B); 33.5 min, 5% acetonitrile (B); 33.5-35 min, 5% acetonitrile (B).

[0027] 1.2 Mass spectrometry ion source parameters: Capillary voltage: Positive: 3kV; Negative: 2.5kV.

[0028] Sample cone hole voltage: 40V.

[0029] Source offset voltage: 80V.

[0030] Ion source temperature: 120℃.

[0031] Desolventization temperature: ±500℃, ±400℃.

[0032] Desolventization flow rate: +1000 L / H, -800 L / H.

[0033] Atomizing gas pressure: 6.5 Bar.

[0034] 1.3 Mass Spectrometry Methods Electrospray ionization (ESI) source, scanning in positive and negative ion modes separately, MS E Continue full scan mode, scan time 0.2 s, scan range 50–1200. In MS... E Collision energies were used, with low collision energies at 6 V and high collision energies at 15-45 V. Sodium formate was used for mass spectrometry calibration, and leucine enkephalin (positive ion mode m / z 556.2771, negative ion mode m / z 554.2615) was used for real-time mass calibration.

[0035] 1.4 Mass Spectrometry Data Analysis Using the Waters full-process, the raw data were preprocessed using the UNIFI system. The raw data were imported into Masslynx software, and various parameters such as quality error, RT alignment tolerance, and peak width were set. A small molecule library corresponding to the single herbs contained in the Tangshen formula was selected, and a data analysis method was established. After running the program, the extracted data were analyzed to obtain candidate active small molecule components (Table 1). Figure 2 ).

[0036] Table 1. Candidate active small molecule components for kidney tonification, symptom elimination, and wind dispelling. Example 2: Identification of candidate active small molecules in the Kidney-Nourishing, Mass-Eliminating, and Wind-Dispelling Formula Medicines and reagents: Kidney-tonifying, mass-eliminating, and wind-dispelling powder.

[0037] Waters Xevo TQ-XS Triple Quadrupole Liquid Chromatography-Mass Analyzer, Waters Corporation, USA; Merck Milli-Q IQ 7000 ultrapure water system, Merck GmbH; Mangiferin reference standard (content 98%, batch number: N08IB231271, Shanghai Yuanye Biotechnology Co., Ltd.); Verbena isoflavone reference standard (content 98%, batch number: KB383401, Shanghai Yuanye Biotechnology Co., Ltd.); Strychnoside reference standard (content 98%, batch number: JB254124, Chengdu Maidesheng Technology Co., Ltd.); Donkey phytosterol reference standard (content 98%, batch number: GN10421, Shanghai Yuanye Biotechnology Co., Ltd.); Kaempferol reference standard (content 98%, batch number: KB372599, Shanghai Yuanye Biotechnology Co., Ltd.); Quercetin reference standard (content 98%, batch number: JB261209, Shanghai Yuanye Biotechnology Co., Ltd.); Panax notoginsenoside R1 reference standard (content 98%, batch number: JB335638, Shanghai Yuanye Biotechnology Co., Ltd.); Acetonitrile, chromatographic grade, Merck.

[0038] Formic acid, chromatographic grade, Shanghai Aladdin Biochemical Technology Co., Ltd.

[0039] The steps for determining the candidate active ingredients of Yishen Xiaozheng Qufeng Formula by UPLC-Q-TOF / MS include: (1) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the packing material; 0.1% formic acid water and acetonitrile were used as the mobile phase; evaporative light scattering detector was used for detection; only samples could be injected after the suitability was determined to be qualified. (2) Preparation of reference solution: Prepare a 50% (v / v) methanol solution as the reference solution; (3) Preparation of test solution: Take the powder of Yishen Xiaozheng Qufeng Fang, put it in a 10 mL test tube, vortex for 5 min, then sonicate for 30 min, then centrifuge to take the supernatant, filter it through a 0.22 μm filter membrane and bottle it to obtain the test sample; (4) Determination of active ingredients: First, run the reference solution to confirm the baseline. Then, accurately pipette the reference solution and the test solution and inject them into the liquid chromatograph. The injection volume is 2L, the column temperature is 35℃, the sample chamber temperature is 10℃, and the mass accuracy is 0.2ppm. Then run the program to determine the active ingredients.

[0040] 1) Chromatographic conditions and system suitability test 1.1 Liquid Chromatography Conditions Column: Waters ACQUITY TM Premier BEH C18 1.7 µm, 2.1×100 mm; Flow rate: 0.3 mL / min; Column temperature: 35℃; Injection volume: 1 μL; Mobile phase: 0.1% formic acid water (A) and pure acetonitrile (B), gradient elution.

[0041] 35-minute gradient elution program: 0-0.2 min, 5%B; 0.2-5 min, 5%-100%B; 5-6 min, 100%B; 6-6.5 min, 100%-5%B; 6.5-8 min, 5%B.

[0042] 1.2 Mass Spectrometry Sources and Methods Ion source: Electrospray ionization (ESI); Scanning mode: Positive or negative ion mode; Monitoring mode: Multiple reaction detection (MRM); Capillary voltage: 2.5kV; Ion source temperature: 150℃; Desolvation gas temperature: 450℃; Desolvation gas flow rate: 1000L / h; Nebulizing gas: Nitrogen; Collider: Argon.

[0043] 1.3 Mass Spectrometry Data Analysis When using a triple quadrupole detector to identify small molecules, the precursor ion of the target molecule is first selected in Q1 to ensure specificity of the detection. Then, in Q2, collision-induced dissociation (CID) is used to fragment the precursor ion into characteristic fragment ions, which are then screened and monitored in Q3. By comparing the mass-to-charge ratio (MRM channel) and retention time of the precursor ion and daughter ion pairs with reference standards, accurate qualitative and quantitative analysis of small molecules can be achieved (Table 2). Figure 3-5 ).

[0044] Table 2 Mass Spectrometry Information of Small Molecule Monomers of Traditional Chinese Medicine This invention discloses a method for detecting the effective components of Yishen Xiaozheng Qufeng Formula based on UPLC-Q-TOF / MS and ACQUITY Premier / Xevo TQ-XS, UPLC-TQ-MS / MS, belonging to the field of quality control and pharmacodynamic material basis research of traditional Chinese medicine compound formulas.

[0045] This method uses Yishen Xiaozheng Qufeng Formula as the research object. An ethanol / water extraction system was used to obtain a concentrated extract. The chemical components of the formula were systematically qualitatively analyzed by UPLC-Q-TOF / MS, establishing chemical component spectra and characteristic fingerprints. Subsequently, based on the UPLC-TQ-MS / MS platform, the main active ingredients were quantitatively detected and validated in multiple reaction monitoring (MRM) mode. Through the complementary application of two mass spectrometry techniques, a systematic study of the formula's components was achieved, from comprehensive qualitative analysis to precise quantification and methodological validation.

[0046] Method validation results showed that the precision RSD was <2.5%, the repeatability RSD was <3.0%, the spiked recovery rate was 98.5%–102.3%, the linear correlation coefficient R² > 0.995, and the limit of detection was 0.3–0.5 ng / mL. These results indicate that the method has good linearity, precision, stability, and sensitivity, and can achieve accurate quantification and repeatable detection of complex components in traditional Chinese medicine compound prescriptions.

[0047] For those skilled in the art, various other corresponding changes and modifications can be made based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of this invention.

Claims

1. A qualitative and quantitative detection method for the effective components in a kidney-tonifying, mass-eliminating, and wind-dispelling formula, characterized in that, The chemical components of Yishen Xiaozheng Qufeng Formula were systematically identified and quantitatively analyzed using UPLC-Q-TOF / MS and UPLC-TQ / MS combined techniques, including the following steps: (1) Sample pretreatment; (2) Qualitative analysis of the samples was performed using UPLC-Q-TOF / MS; (3) UPLC-TQ / MS was used to perform quantitative analysis of the target components using multiple reaction monitoring; (4) Process the obtained data, determine the content of components, and establish a quantitative basis for active substances.

2. The qualitative and quantitative detection method for the effective components in the Kidney-Nourishing, Symptom-Eliminating, and Wind-Dispelling Formula according to claim 1, characterized in that, The sample pretreatment steps include: weighing 0.5 g of Yishen Xiaozheng Qufeng Fang powder, adding 10 mL of 50% methanol solution, ultrasonically extracting for 30 minutes, filtering, centrifuging to obtain the supernatant, filtering through a 0.22 μm microporous membrane to obtain the sample solution for mass spectrometry analysis.

3. The qualitative and quantitative detection method for the effective components in the Kidney-Nourishing, Symptom-Eliminating, and Wind-Dispelling Formula according to claim 1, characterized in that, The mobile phase used in the UPLC chromatographic analysis consisted of 0.1% formic acid water and acetonitrile, with the following gradient elution program: 0–2 min 5%B, 2–30 min 5%→95%B, 30–33 min 95%B, 33–35 min 95%→5%B; the flow rate was 0.3 mL / min, and the column temperature was 35 ℃.

4. The qualitative and quantitative detection method for the effective components in the kidney-tonifying, phlegm-reducing, and wind-dispelling formula according to claim 1, characterized in that, The UPLC-Q-TOF / MS uses an electrospray ionization source, a bipolar mode of positive and negative ions, a low collision energy of 6 eV, a high collision energy of 15-45 eV, and a mass detection accuracy of ≤0.2 ppm.

5. The qualitative and quantitative detection method for the effective components in the Kidney-Nourishing, Symptom-Eliminating, and Wind-Dispelling Formula according to claim 1, characterized in that, The UPLC-TQ / MS uses multiple reaction monitoring mode for quantitative detection, with the following parameters: capillary voltage 2.5 kV, ion source temperature 150 ℃, desolvation temperature 450 ℃, nebulizing gas nitrogen, and collision gas argon.

6. The qualitative and quantitative detection method for the effective components in the Kidney-Nourishing, Symptom-Eliminating, and Wind-Dispelling Formula according to claim 1, characterized in that, The main active ingredients detected by the method include one or more of the following compounds: piperine, dihydroquercetin, cholesterol or cholesterol compounds, clofentin, strychnine, 6,8-di-C-α-L-arabinose apigenin, verrucoside, 9-hexadecenoic acid, tristyric acid, δ-tocopherol, 3,9-di-O-methylnisorline, strychnine, loganin, and quercetin.

7. The qualitative and quantitative detection method for the effective components in the Kidney-Nourishing, Symptom-Eliminating, and Wind-Dispelling Formula according to claim 1, characterized in that, The UPLC-TQ / MS system employs a triple quadrupole mass spectrometry system. In multiple reaction monitoring mode, it achieves accurate qualitative and quantitative analysis of small molecule components through parent ion screening, collision-induced dissociation, and daughter ion monitoring. The detected active ingredients include gentianin, verbascoside, loganin, gentianol, kaempferol, quercetin, and notoginsenoside R1. Their parent-daughter ion pairs and retention times are consistent with those of the reference standard.

8. The qualitative and quantitative detection method for the effective components in the Kidney-Nourishing, Symptom-Eliminating, and Wind-Dispelling Formula according to claim 1, characterized in that, The method described above is applied to the quality control of Yishen Xiaozheng Qufeng Formula and its pharmacodynamic material basis research in the prevention and treatment of diabetic nephropathy.

9. The qualitative and quantitative detection method for the effective components in the Kidney-Nourishing, Symptom-Eliminating, and Wind-Dispelling Formula according to claim 1, characterized in that, The method is applied in the detection of traditional Chinese medicine compound formulas containing ingredients such as Astragalus membranaceus, Panax notoginseng, Euryale ferox, Lonicera japonica, Spatholobus suberectus, Sparganium stoloniferum, or Dioscorea opposita.

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