Elucidation of the Material Basis of Yiqi Qingjie Formula Against IgA Nephropathy Using UHPLC-Q-Orbitrap HRMS Integrated with Network Pharmacology

Our research identifies bioactive compounds of Yiqi Qingjie formula, a traditional Chinese total compound, treating IGA nephropathy, using HIMS serum, pharmaco chemistry, and network pharmacology. Existing methods rely on predictive compounds rather than absorbed one. This protocol first experimentally verified blood absorptive constitution.

To begin, obtain the prepared Yiqi Qingjie, concentrated herbal extract, and methanolic reference standard solutions. Pipette 100 microliters of the Yiqi Qingjie aqueous extract into a 1.5 milliliter centrifuge tube. Add 300 microliters methanol to the tube and vortex for one minute.

Centrifuge the mixture at 1, 800 G and four degrees Celsius for 10 minutes. Confirm that the resulting supernatant is clear and free of suspended particles. Add 100 microliters of the supernatant to 100 microliters ultrapure water.

Vortex gently to mix, and transfer the solution into an auto sampler vial. Obtain frozen, hemolysis free serum samples collected from terminally anesthetized sprague dolly rats. After controlled dosing with Yiqi Qingjie extract, pipette 100 microliters of serum into a 1.5 milliliter centrifuge tube.

Add 300 microliters of methanol and vortex for 10 minutes. Centrifuge the mixture at 1, 800 G and four degrees Celsius for 10 minutes. Collect 270 microliters of the supernatant and transfer it to a clean tube.

Use a vacuum centrifugal concentrator, set at 120 G and 37 degrees Celsius for four hours to dry the supernatant completely. Add 90 microliters of 50%methanol aqueous solution to the residue vortex for one minute to dissolve the residue completely and centrifuge at 1, 800 G and four degrees Celsius for 10 minutes. Collect 80 microliters of the supernatant and transfer it into an auto-sampler vial.

Switch on and initiate the ultra high performance, liquid chromatography, or UHPLC system, equipped with a chromatographic column. Keep the binary mobile phase ready with phase A as water containing 0.1%formic acid and phase B as aceto nitryl. Apply a gradient elution program at a flow rate of 0.3 milliliters per minute.

Set the column temperature to 40 degrees celsius and the injection volume to 6.0 microliters. Next, switch on and initiate a quadruple orbitrap mass spectrometer equipped with a heated electro spray ionization spray probe. Set the resolution to 17, 500, auto gain control target as one times 10 to the power of five, maximum isolation time 50 milliseconds, and select up to 10 most intense parent ions with dynamic exclusion.

Then set the isolation window of mass to charge to two, collision energy of 10 volts, 30 volts and 60 volts, and intensity threshold one times 10 to the power of five. Set the ionization voltage to 3.7 kilovolts in positive mode or 3.5 kilovolts in negative mode, capillary temperature to 320 degrees Celsius, sheath gas pressure to 30 pounds per square inch, auxiliary gas pressure to 10 pounds per square inch, desolvation temperature to 300 degrees Celsius, and use nitrogen as sheath, auxiliary, and collision gas at 1.5 millitorr. Set full scan parameters with resolution 70, 000, auto gain control target one multiplied by 10 to the power of six, maximum isolation time 50 milliseconds, and mass to charge scan range 100 to 1, 500.

Acquire data in full scan data dependent, tandem mass spectrometry mode. Use specialized data processing software to process the acquired mass spectrometry data. Navigate to the import data module and upload raw data files.

Execute the peak picking module and perform peak deconvolution to group adduct ions and generate compound features. Search reference compound databases, target prediction databases, and relevant literature for candidate identification. Compare experimental data with reference standards to confirm matches.

Accept compound identification only when mass accuracy is within plus or minus five parts per million. Spectral similarity is at least 0.85. Isotopic matching is at least 90%and retention time deviation is less than 0.2 minutes.

Annotate the identified compounds as MSI level one. Use accurate mass measurements and computational spectral prediction to perform putative identification for compounds without reference standards. Accept identifications when precursor ion mass error is within plus or minus five parts per million, fragment ion mass error within plus or minus 10 parts per million, spectral similarity is at least 0.60.

Isotopic matching is at least 90%and retention time deviation is less than 1.0 minute. Annotate the putatively identified compounds as MSI level two. The identified compounds are further investigated, using network pharmacology and molecular docking to support downstream interpretation and results.

The base peak ion chromatogram of the Yiqi Qingjie aqueous extract was obtained in positive ion mode and in negative ion mode. In the PPI network, node size and color varied with parameter values. TP53, SRC, AKT1, ESR1, and STAT3 ranked among the top five core targets.

The molecular docking binding energy heat map presented the binding energies between core targets, and key blood absorbed compounds with ligands represented along the vertical axis and receptor proteins along the horizontal axis. This protocol identifies orally absorbed total compound in blood and predict the therapeutic targets and potential biological pathways. A curative compound identification, using this protocol request high quality data, proper serum sample times, and careful sample preparation.

Future studies convey that predictive compound target interactions and explore molecular mechanism of each Yiqi Qingjie formula in IgA nephropathy.