Development of a three-step-based novel strategy integrating DMPK with network pharmacology and bioactivity evaluation for the discovery of Q-markers of traditional Chinese medicine prescriptions: Danlou tablet as an example.

BACKGROUND: Quality marker (Q-marker) serves an important role in promoting the standardization of the quality of traditional Chinese medicine (TCM) prescriptions. However, discovering comprehensive and representative Q-markers from TCM prescriptions composed of multiple components remains difficult. PURPOSE: A three-step-based novel strategy integrating drug metabolism and pharmacokinetics (DMPK) with network pharmacology and bioactivity evaluation was proposed to discover the Q-markers and applied to a research example of Danlou tablet (DLT), a famous TCM prescription with remarkable and reliable clinical effects for coronary heart disease (CHD). METHODS: Firstly, the metabolic profile in vivo of DLT was systemically characterized, and the pharmacokinetic (PK) properties of PK markers were then investigated. Secondly, an integrated network of "PK markers - CHD targets - pathways - therapeutic effects" was established to screen out the crucial PK markers of DLT against CHD. Thirdly, the crucial PK markers that could exhibit strong myocardial protection activity in the H9c2 cardiomyocyte model were selected as the candidate Q-markers of DLT. According to the proportion of their Cmax value in vivo, the candidate Q-markers were configured into a composition; the bioactivity was then evaluated to confirm their synergistic effect and justify their usage as Q-markers. RESULTS: First of all, a total of 110 DLT-related xenobiotics (35 prototypes and 75 metabolites) were detected in bio-samples, and the pharmacokinetic properties of 13 PK markers of DLT were successfully characterized, revealing the quality transitivity and traceability from prescription to in vivo. Then, 6 crucial PK markers with three topological features (degree, betweenness, and closeness) greater than the average values in the pharmacology network were screened out as the key components of DLT against CHD. Furthermore, among these 6 crucial PK markers, 5 components (puerarin, alisol A, daidzein, paeoniflorin, and tanshinone IIA) with strong myocardial protection activity were chosen as the candidate Q-markers to constitute a new composition. The composition activated the expression of the PI3K/AKT pathway and exhibited strong myocardial protection activity, and the effective concentrations (nM level) of these components in the composition were significantly lower than their individually effective concentrations (µM level), indicating that there was a certain synergistic effect between them. Hence, the 5 components with multiple properties, including testability, quality transitivity and traceability from prescription to in vivo, effectiveness, and compatibility contribution, were defined as comprehensive and representative Q-markers of DLT. CONCLUSION: This study not only presented a novel idea for the revelation of comprehensive and representative Q-markers in quality control research of TCM prescriptions, but also identified the reasonable Q-markers of DLT for the first time to improve the quality control level of DLT.

Qualitative and quantitative analysis of the chemical profile for Gualou-Xiebai-Banxia decoction, a classical traditional Chinese medicine formula for the treatment of coronary heart disease, by UPLC-Q/TOF-MS combined with chemometric analysis.

Gualou-Xiebai-Banxia decoction (GXB) is one of the famous classical traditional Chinese Medicine (TCM) formula for the treatment of chest stuffiness and pains syndrome in Chinese medicine, i.e., coronary heart disease (CHD) in modern medicine. Being compared with Gualou-Xiebai Baijiu-decoction which only consists of Trichosanthis Pericarpium (TP), Allii Macrostemonis Bulbus (AMB) and wine, GXB is composed of another one additional herbal medicine, Pinellinae Rhizoma Praeparatum (PRP), and is more suitable to treat severe atherosclerosis and dyslipidemia. However, the comprehensive chemical composition of GXB is still unclear, which has seriously hindered the discovery of its effective components for improving the clinical symptoms of CHD. The present study aimed to investigate the overall chemical profile of GXB qualitatively and quantitatively by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS), and further explore the chemical contribution of PRP to this formula combined with chemometric approach. First, a total of 151 components, including steroidal saponins, flavonoids, triterpenoids, nitrogenous and other types components, were detected and characterized by UPLC-Q/TOF-MS in GXB. Then, flavonoids and nitrogenous could be qualitatively observed enrichment in GXB compared to those in GXB-dePRP (GXB deducted PRP in the formula). Furthermore, 19 characteristic components were selected for quantitative comparison between GXB and GXB-dePRP by UPLC-MS/MS combined with chemometric method. These findings indicated that steroidal saponins were the most abundant components in GXB, while the introduction of PRP could not only enrich the structural types of chemical compounds in this formula, but also increase the abundance of active components from other composed herbal medicines, TP and AMB. Taken together, this study developed and validated sensitive and practical methods for qualitative and quantitative analysis of GXB, and clarified the chemical contribution of PRP to this formula. These results laid a solid chemical foundation for further in vivo disposal investigation to screen out the potential effective components as well as therapeutic mechanism research of GXB.

The discovery of Q-markers of Qiliqiangxin Capsule, a traditional Chinese medicine prescription in the treatment of chronic heart failure, based on a novel strategy of multi-dimensional "radar chart" mode evaluation.

BACKGROUND: Qiliqiangxin Capsule (QLQX), a traditional Chinese medicine (TCM) prescription, is especially used for clinical treatment of chronic heart failure (CHF) in China. However, the holistic quality control of QLQX has not been well established due to lack of system research on the quality marker (Q-marker). PURPOSE: In this study, a new strategy of multi-dimensional "radar chart" mode was proposed to overcome the problem that traditional methods cannot evaluate the multiple properties of Q-markers comprehensively and visually, and the strategy was successfully applied to discover the Q-markers of QLQX. METHODS: First, nineteen prototypes that entered the in vivo systemic circulation were selected out as the candidate Q-markers based on our previous studies of chemical and in vivo metabolic profiles. Then, their contents in QLQX were quantitatively analyzed by UHPLC-MS/MS, and the bioactivities on the H9c2 cardiomyocytes cell model was evaluated. The network of in vivo component-target closely related to CHF was further constructed. Finally, a multi-dimensional "radar chart" mode was developed and corresponding Regression Area (RA) and Coefficient Variation (CV) were calculated after data standardization and integration visually based on the Q-marker related multiple characteristics (including the compatibility contribution of herbal medicines, the content, the bioactivity, the in vivo predicted bioavailability and the degree of network pharmacology of candidate components in the TCM prescription). RESULTS: By comparison of RA and CV of the chemicals in the "radar chart", seven compounds mainly from King and Minister herbs (songorin, calycosin-7-O-ß-D-glucopyranoside, astragaloside, tanshinone IIA, ginsenoside Re, hesperidin and alisol A) were screened out as the Q-markers of QLQX, showing the reasonable compatibility contribution and high content in QLQX, preferable pharmacological effect on CHF, as well as good bioavailable characteristics and high target hits in system pharmacology. CONCLUSION: The Q-marker discovery of QLQX in this study laid an important foundation for its quality control improvement, and the mode standardized the abstract definitions of Q-marker and realized the comprehensive assessment of multiple properties of Q-marker in TCM prescriptions, which has a reference value for revealing the Q-marker in the quality control researches of TCM prescriptions.

Characterization of chemical profile and quantification of representative components of DanLou tablet, a traditional Chinese medicine prescription, by UHPLC-Q/TOF-MS combined with UHPLC-TQ-MS.

DanLou tablet (DLT), a famous traditional Chinese medicine prescription (TCMP) consisting of 10 herbal medicines, is extensively used for the treatment of angina pectoris and acute coronary syndrome in China. However, active chemical constituents responsible for the therapeutic effects still remain unclear, due to the fact that the complex composition in DLT have not been holistically clarified. Therefore, this study aimed to characterize the chemical profile and simultaneously quantify the representative components in DLT. First, 157 chemical constituents including flavonoids, triterpenoids, tanshinones, lactones, phenolic acids, paeoniflorins and the other types of components were detected, among which 39 were exactly identified by comparing their retention times and MS fragmentation behaviors with those of authentic standards by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q/TOF-MS). Moreover, 33 representative components were simultaneously quantified by ultra-high performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-TQ-MS), which were selected based on following three principles: qualitative and quantitative markers in the Chinese Pharmacopeia (2015 edition), bioactive components possessing cardiovascular-related in vivo or in vitro activities and those derived from 10 consisted herbs in DLT with a diversity of representative structure types. The method was validated in terms of linearity, precision, repeatability and recovery and successfully applied for the quality evaluation of 20 batches of DLT samples. Further chemometric analysis indicated that danshensu and salvianolic acid B were the most significant quantitative markers for the content fluctuation of DLT. In summary, the chemical profiles of DLT were systematically characterized and a practical quantitative method combined with chemometrics was developed to evaluate the intrinsic quality of multiple DLT samples in this study. The present work would be helpful for guaranteeing the safety, efficacy, and controllability in clinical medication of DLT.

[Corrigendum] The role and underlying mechanisms of microRNA­214 in Legg­Calvé­Perthes disease.

Following the publication of this paper, the authors have realized that the name of their institute, as presented in the affiliations, should have been changed from "Children's Hospital Affiliated to Nanjing Medical University" to "Children's Hospital of Nanjing Medical University". Therefore, the authors' affiliations, and the affliation address, should have appeared as follows: Hui Zhu*, Xinyue Qi*, Yuehe Liu*, Wei Liao, Xiangshui Sun and Yuping Tang. Department of Orthopedics, Children's Hospital of Nanjing Medical University, Nanjing, Jiangsu 210009, P.R. China. *Contributed equally. The authors confirm that there are no further errors in the study, and all the authors agree to this correction. The authors regret this error in the affiliations, and apologize for any inconvenience caused. [the original article was published in Molecular Medicine Reports 20: 685­692, 2019; DOI: 10.3892/mmr.2019.10271].

The role and underlying mechanisms of microRNA­214 in Legg­Calvé­Perthes disease.

Legg­Calvé­Perthes disease (LCPD) is a pediatric form of femoral head osteonecrosis with unknown etiology. MicroRNAs (miRs) have been revealed to serve an important role in LCPD. MiR­214 serves an important role in chondrogenesis. The aim of the present study was to investigate the potential role of miR­214 in LCPD and the underlying mechanisms. The expression levels of miR­214 and B­cell lymphoma 2 (Bcl­2)­associated X protein (Bax) in dexamethasone (DEX)­treated TC28 cells, and the femoral head cartilage tissues, serum and primary chondrocytes of patients with LCPD, and healthy individuals were determined via reverse transcription quantitative polymerase chain reaction and western blot analysis. A luciferase reporter assay was conducted to investigate the association between miR­214 and Bax, while cell viability was determined via an MTT assay, and flow cytometry was performed to investigate cell apoptosis. The results revealed that miR­214 was downregulated and Bax was upregulated in DEX­treated TC28 cells and tissues obtained from patients with LCPD. MiR­214 was demonstrated to directly target Bax and negatively regulate its expression. DEX administration significantly suppressed cell proliferation, promoted apoptosis and decreased the Bcl­2/Bax ratio in TC28 cells; overexpression of miR­214 induced opposing effects, which were reversed by Bax overexpression. In conclusion, the results indicated that miR­214 and Bax may be potential therapeutic targets for the future clinical treatment of LCPD.

[Association of IFNG gene polymorphisms with susceptibility to preeclampsia among pregnant woman from Shaanxi].

OBJECTIVE: To assess the association of IFNG gene polymorphisms with preeclampsia among pregnant woman from Shaanxi Province. METHODS: Genomic DNA was extracted from peripheral blood samples collected from 280 patients with preeclampsia and 344 healthy pregnant women. Five tag single nucleotide polymorphisms (SNPs) of the IFNG gene (rs2069705, rs2430561, rs1861493, rs2069718, and rs2193050) were genotyped with a SNaPshot method. Genotypic and allelic frequencies were evaluated with a Chi square test. Genotype data was corrected by Logistic regression for body mass index and age. The level of IFN-gamma was determined with an ELISA assay. RESULTS: The distribution of five tag SNPs all conformed to Hardy-Weinberg equilibrium (P> 0.05). Significant association with preeclampsia was found with the T allele of rs2430561 (OR=1.54, 95% CI:1.15-2.09, P=6.99× 10-3), under a dominant model (OR=3.77, 95% CI: 1.09-13.29, P=0.029) and a recessive model (OR=1.53, 95% CI:1.09-2.15, P=0.018). For the patient group, the IFN-gamma level of those with a TT genotype for rs2430561 was significantly higher than those with an AA or AT genotype [(13.69± 0.79) pg/mL vs. (13.11± 1.56) pg/mL, P< 0.05]. CONCLUSION: Polymorphism of the rs2430561 locus of the IFNG gene is associated with increased risk for preeclampsia as well as serum level of IFN-gamma among pregnant woman from Shaanxi. The role of the IFNG gene in the regulation of preeclampsia requires further investigation.