Shineway Pharmaceutical Group Co. Ltd.

Ischemic stroke is caused by local lesions of the central nervous system and is a severe cerebrovascular disease. A traditional Chinese medicine, Yiqi Tongluo Granule (YQTL), shows valuable therapeutic effects. However, the substances and mechanisms remain unclear.

We combined network pharmacology, multi-omics, and molecular biology to elucidate the mechanisms by which YQTL protects against CIRI.

We innovatively created a combined strategy of network pharmacology, transcriptomics, proteomics and molecular biology to study the active ingredients and mechanisms of YQTL. We performed a network pharmacology study of active ingredients absorbed by the brain to explore the targets, biological processes and pathways of YQTL against CIRI. We also conducted further mechanistic analyses at the gene and protein levels using transcriptomics, proteomics, and molecular biology techniques.

YQTL significantly decreased the infarction volume percentage and improved the neurological function of mice with CIRI, inhibited hippocampal neuronal death, and suppressed apoptosis. Fifteen active ingredients of YQTL were detected in the brains of rats. Network pharmacology combined with multi-omics revealed that the 15 ingredients regulated 19 pathways via 82 targets. Further analysis suggested that YQTL protected against CIRI via the PI3K-Akt signaling pathway, MAPK signaling pathway, and cAMP signaling pathway.

We confirmed that YQTL protected against CIRI by inhibiting nerve cell apoptosis enhanced by the PI3K-Akt signaling pathway.

Qinggusan is the 69th prescription in the first batch of "Catalogue of Ancient Chinese Classic Formulas". In modern clinical practice， Qinggusan is mainly used to treat noninfectious fever. However， because few studies on Qinggusan reference samples and their quality value transfer are available， the development and promotion of its compound preparations are restricted. Therefore， establishing an accurate and comprehensive quality control method to clarify the critical quality attributes of Qinggusan reference samples is of great importance. In this study， 15 batches of Qinggusan reference samples were processed to determine the range of their dry extract ratios. Quantitative high-performance liquid chromatography （HPLC） fingerprint analysis was performed using a Waters Symmetry Shield RP18 column （250 mm×4.6 mm， 5 μm） with acetonitrile-0.1% （v/v） formic acid aqueous solution as the mobile phase in gradient elution mode. The flow rate was 1.0 mL/min， the column temperature was 30 ℃， and the detection wavelength was 254 nm. The HPLC fingerprints of the Qinggusan reference samples were established under these conditions to evaluate their similarity. The established method was systematically validated and found to demonstrate good precision， repeatability， and sample stability. Subsequently， characteristic peaks were identified and attributed by HPLC-quadrupole-time-of-flight-mass spectrometry （HPLC-Q-TOF-MS） analysis. MS was performed in electrospray ionization mode， the data were collected in both positive- and negative-ion modes， and the detection range was m/z 50-2000. The contents and transfer rate ranges of the index components， namely， gentiopicrin， mangiferin， picroside Ⅱ， picroside Ⅰ， and glycyrrhizic acid， were determined to analyze the quality value transfer of the samples. The results demonstrated that the dry extract rate of the 15 batches of Qinggusan reference samples ranged from 24.10% to 26.88% and that their fingerprint similarities were generally greater than 0.95. Twelve common peaks were identified by reference identification， literature comparison， and high-resolution MS analysis. Twelve compounds， including six iridoid glycosides， two flavonoids， one alkaloid， one triterpenoid saponin， and two others. Among them， L-picein， androsin， picroside Ⅳ， picroside Ⅱ and picroside Ⅰ were from Picrorhizae Rhizoma， loganin acid， swertiamarin and gentiopicrin were from Gentianae Macrophyllae Radix， neomangiferin and mangiferin were from Anemarrhenae Rhizoma， dichotomine B was from Stellariae Radix， and glycyrrhizic acid was from Glycyrrhizae Radix et Rhizoma. The five key components presented good linear relationships in their respective linear ranges， and all correlation coefficients were higher than 0.999. The relative standard deviations （RSDs） of precision， stability， and repeatability were less than 1.3%. The average recoveries varied between 95.92% and 102.5%， with RSDs less than 3.9%； these values meet the requirements defined in the 2020 edition of the Chinese Pharmacopoeia. The contents of gentiopicrin， mangiferin， picroside Ⅱ， picroside Ⅰ， and glycyrrhizic acid in the 15 batches of reference samples were in the range of 17.92-27.55， 1.83-4.42， 23.08-36.44， 8.43-15.04， and 0.94-2.39 mg/g， respectively， and their transfer rates from the decoction pieces to the reference samples were 47.91%-63.95%， 22.96%-59.39%， 60.82%-77.82%， 64.25%-99.53%， and 15.30%-39.30%， respectively. In this study， the chemical components of Qinggusan reference samples were comprehensively identified and their quality value transfer was studied through the combination of HPLC fingerprinting and MS. Clarification of the critical quality attributes of Qinggusan reference samples could provide a basis for the quality control of Qinggusan compound preparations.

Chinese rice wine (CRW) is a well-known drink and functional food that is used in traditional Chinese medicine. However, there is still a lack of quality control and evaluation methods for CRWs.

The study aimed to establish a new method that can serve both as quality control and evaluation method and, as well as an identification method for CRWs.

Compound identification in different CRW samples and determination of uracil, xanthine, uridine, adenine, guanosine, 5-hydroxymethylfurfural, and adenosine contents from 29 CRW samples from 14 brands were performed using UPLC-PDA/TOF-MS. The dual-column chromatographic separation of CRW was performed using CORTECS T3 coupled to HSS T3. The optimal mobile phase consisted of water with 0.1% formic acid, 40 mM ammonium acetate, and methanol: acetonitrile (2:1). Furthermore, to compare the UPLC fingerprints between CRWs of different brands, a similarity analysis was performed to classify the CRW samples. Finally, network pharmacology and in vitro efficacy and toxicity tests were used to investigate the biological function of the seven components and CRWs.

A total of 55 compounds were unambiguously or tentatively identified. Among them, nucleoside, pyrimidines and purines were reported in CRW for the first time. The seven components were successfully determined, and their contents showed large variations among different brands of CRW, which was consistent with the results of the chromatographic fingerprint similarities. The results of in vitro efficacy and toxicity tests indicated that CRWs and seven components had obvious protective effect on H9c2 cell injury induced by the H₂O₂ model. Network pharmacology analysis showed that these seven compounds might be the main active components of CRW that promote blood circulation and ventilation.

This study revealed that dual-column chromatographic separation is an effective method for quantitative and chromatographic fingerprint analyzes of complex samples, and seven compounds can be used for the quality evaluation and control of CRWs.