Identification and quality evaluation of different processed products of Aconitum carmichaelii by ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry and ultra-high-performance liquid chromatography-tandem mass spectrometry.

Aconitum carmichaelii is widely used to treat chronic and intractable diseases due to its remarkable curative effect, but it is also a highly toxic herb with severe cardiac and neurotoxicity. It has been combined with honey for thousands of years to reduce toxicity and enhance efficacy, but there has been no study on the chemical constituent changes in the honey-processing so far. In this study, the chemical constituents of A. carmichaelii before and after honey-processing were characterized by ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry. The results showed that a total of 118 compounds were identified, of which six compounds disappeared and five compounds were newly produced after honey-processing, and the cleavage pathway of main components was elucidated. At the same time, 25 compounds were found to have significant effects on different products, among which four compounds with the biggest difference were selected for quantitative analysis by ultra-high-performance liquid chromatography-tandem mass spectrometry. This study not only explained the chemical differences between the different products, but also helped to control the quality of the honey-processed products more effectively, and laid a foundation for further elucidating the mechanism of chemical constituent change during the honey-processing of A. carmichaelii.

Guizhi Fuling Capsule inhibits uterine fibroids growth by modulating Med12-mediated Wnt/ß-Catenin signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Guizhi Fuling Capsule (GFC) is a famous traditional Chinese medicine (TCM) formula recorded in Synopsis of the Golden Chamber, which has achieved obvious effects in the treatment of uterine fibroids (UFs). AIM OF STUDY: Mediator complex subunit 12 (Med12) mutations were closely related to UFs in 85% of fibroid cases. The Wnt/ß-Catenin signaling pathway plays an important role in the occurrence and development of UFs. This study aims to explore the pharmacological mechanism of GFC against UFs in which the Med12-mediated Wnt/ß-Catenin pathway is involved. MATERIALS AND METHODS: Med12 was silenced in uterine fibroid cells (UFCs) using a lentivirus-based Med12 gene-specific RNA interference (RNAi) strategy. Cell proliferation was performed by CCK-8 assay, cell apoptosis and cell cycle were measured by flow cytometry. The rat model of UFs was established by injecting estradiol benzoate and progesterone. Forty-eight rats were divided into six groups, the low-dose GFC (L-GFC) group, the medium-dose GFC (M-GFC) group and the high-dose GFC (H-GFC) group were intragastrically treated with GFC solution at 0.25 g/kg, 0.50 g/kg and 1.00 g/kg per day for 8 weeks, the positive control (PC) group was administrated with mifepristone (2.70 mg/kg/day), the normal control (NC) group and the model control (MC) group were given equal volume of normal saline once a day for 8 weeks. The histopathological changes of uterine tissues were evaluated by H&E staining. The expression of Med12 in uterine tissues were detected by immunohistochemistry. The protein and mRNA levels of associated genes were evaluated by western bolt and real time-PCR, respectively. Related indicators involved in Wnt/ß-Catenin pathway, such as Wnt1, ß-Catenin, Cyclin D1, TCF1/TCF7 and C-myc, were compared among different groups. RESULTS: The Wnt/ß-Catenin signaling pathway was inhibited after Med12 gene was knocked out in UFCs. GFC-containing serum could induce cell apoptosis, make the cell cycle stagnated in G0/G1 phase to inhibiting the proliferation and reduce the expression of Wnt1, ß-Catenin, Cyclin D1, TCF1/TCF7, and C-myc in control-shRNA cells, while had no significant effect on Med12-shRNA cells. Compared with the MC group, the weight, endometrial thickness, and pathological structure of the uterus in the GFC treated groups were significantly improved. The expression of Med12, Wnt1, ß-Catenin, Cyclin D1, TCF1/TCF7, and C-myc that related to Wnt/ß-Catenin pathway in the GFC treated groups were decreased with the increase of dosage administration. CONCLUSIONS: GFC inhibited UFs growth, which was directly associated with Med12 modulated Wnt/ß-Catenin signaling pathway. This study provided new perspective to understand the therapeutic mechanism of UFs.

Effects of Processing Method Changes in Main Volatile Compounds of Qixue Shuangbu Prescription by Needle Trap Device Coupled with Gas Chromatography-Triple Quadrupole Mass Spectrometry.

Qixue Shuangbu Prescription (QSP) has been widely applied in the treatment of chronic heart failure (CHF). Previous clinical studies have found that the efficacy of processed QSP was significantly enhanced in the treatment of CHF. We have identified and analyzed the nonvolatile components before and after processing of QSP, and predicted the mechanism of synergistic effect after processing in the treatment of CHF. However, the synergistic mechanism of processed QSP caused by the difference of volatile components was still unclear. In this study, we developed a method of needle trap device coupled with gas chromatography-triple quadrupole mass spectrometry to elucidate the difference of volatile components between crude and processed QSP. The established method has been used to identify 104 volatile compounds in crude and processed QSP. The results of multivariate data showed 38 differential compounds were screened as potential markers, which would further explain the mechanism of processing synergistic effect of processed QSP. This study successfully developed the method to elucidate its processing mechanism based on the difference of volatile compositions between crude and processed QSP for the first time, and it would provide a novel analytical strategy for the impacts of different processing methods on main volatile compounds in traditional Chinese medicine.

Comparative pharmacokinetics study of six effective components between two dosage forms of Qixue-Shuangbu Prescription in rats by UPLC-MS/MS.

Qixue-Shuangbu Prescription (QSP) is an efficacious prescription for treating heart failure, myocardial ischemia and other diseases. It is composed of nine Chinese herbs. This study investigated and compared the pharmacokinetics of QSP in rats by UPLC-MS/MS between two dosage forms of traditional decoction (TD) and compound tincture (CT). Owing to the complexity of the chemicals in QSP, ginsenoside Rg1, ginsenoside Re, ferulic acid, astragaloside IV, rhein and calycosin were chosen for the pharmacokinetics study. The method established for detecting serum specimens was shown to have acceptable selectivity, linearity, lower limit of quantitation, precision, accuracy, recovery, matrix effect and stability. The peak concentration, AUC0-t and AUC0-∞ of ginsenoside Re, ginsenoside Rg1, ferulic acid and rhein were significantly increased after oral administration of CT (P < 0.05), the half-life of ferulic acid in the CT group was lower than that in the TD group (P < 0.05) and the half-life and AUC0-∞ of astragaloside IV in the CT group were significantly increased (P < 0.05), which revealed that wine-processing could influence the bioavailability and the elimination of these compounds. For better clinical efficacy, we suggest that the CT dosage form of QSP should be selected.

Potential Synergy Mechanism of Processing Methods for the Basic Remedies of Qixue Shuangbu Prescription Based on Integrated Metabolomics Strategy and Network Pharmacology Study.

Qixue Shuangbu Prescription (QSP) is a famous traditional Chinese medicine (TCM) formula widely used for the treatment of chronic heart failure (CHF). Previous clinical studies have found that the efficacy of processed QSP has been significantly enhanced in the treatment of CHF. However, the synergistic mechanisms of processed QSP to enhance the treatment of CHF are still unclear. Generally, the changes in clinical effects mainly result from the variations of inside chemical basis caused by the TCM processing procedure. In this study, we developed a network pharmacology-integrated metabolomics strategy to clarify the difference of the effective compounds between crude and processed QSP, and further explain the mechanism of processed QSP to produce a synergistic effects. As a result, 69 different compounds were successfully screened, identified, quantified and verified as the most potential marker compounds. These different chemical components may play an anti-CHF and enhance the therapeutic effect through 52 action pathways such as estrogen signaling pathway, ubiquitin mediated proteolysis, protein processing in endoplasmic reticulum, etc. This study revealed that the proposed network pharmacology-integrated metabolomics strategy was a powerful tool for explaining the mechanism of synergistic action in the processing of QSP, further controlling the quality and understanding the processing mechanism of TCM formulae.