Deciphering mechanism of Zhishi-Xiebai-Guizhi Decoction against hypoxia/reoxygenation injury in cardiomyocytes by cell metabolomics: Regulation of oxidative stress and energy acquisition.

Myocardial ischemia/reperfusion (MI/R) injury is a life-threatening syndrome with high morbidity and mortality. Zhishi-Xiebai-Guizhi Decoction (ZSXBGZD) is a classic traditional Chinese medicine formula, used to treat cardiovascular diseases for centuries. However, its underlying medicinal mechanism has not been clearly elucidated, which hinders its widespread application. Here, the curative effects and therapeutic mechanism of ZSXBGZD against MI/R were addressed based on an integration of pharmaceutical evaluation and cellular metabolomics. First, a hypoxia/reoxygenation (H/R) model in H9c2 cells was employed to resemble MI/R and multiple pharmacological indicators were performed to assess the efficacy of ZSXBGZD. The results showed that ZSXBGZD possessed exceptional ability in attenuating cardiomyocyte injury, concerning oxidative stress, mitochondrial dysfunction, energy acquisition and cell apoptosis. Furthermore, a cell metabolomics approach based on HILIC and UPLC-Q-TOF-MS coupled with multivariate analysis was conducted to explore the metabolic regulation of ZSXBGZD. 38 differential polar metabolites related to H/R were uncovered, and 34 of them were reversed to normal state after the treatment of ZSXBGZD, revealing the perturbations of energy metabolism and amino acid metabolism. Moreover, formula decomposition justified the combination of single herbs to form ZSXBZGD and confirmed the pivotal status of Allii Macrostemonis Bulbus and Trichosanthis Fructus.

Chemical profiling and quality evaluation of Zhishi-Xiebai-Guizhi Decoction by UPLC-Q-TOF-MS and UPLC fingerprint.

Zhishi-Xiebai-Guizhi Decoction (ZSXBGZD), a traditional Chinese medicine (TCM) formula, has been used for treatment of coronary heart disease and myocardial infarction for nearly two thousand years. However, the chemical composition of ZSXBGZD is still unclear. In order to obtain the chemical profile of ZSXBGZD, an ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) method was utilized for the identification of its multi-constituents. As a result, a total of 148 compounds were identified based on their retention times, accurate masses and MS/MS data. In addition, an optimized UPLC fingerprint analysis, combined with chemometrics such as similarity analysis (SA), hierarchical cluster analysis (HCA), principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) was developed for quality assessment of ZSXBGZD. Multivariate data analysis revealed that samples could be classified correctly according to their geographic origins, and four compounds neohesperidin, naringin, guanosine and adenosine contributed the most to classification. The established UPLC method with multi-wavelength detection was further validated and implemented for simultaneous quantification of 12 representative ingredients in the prescription, including guanosine, adenosine, 2'-deoxyadenoside, syringin, magnoloside A, forsythoside A, naringin, hesperidin, cinnamaldehyde, neohesperidin, honokiol and magnolol. This is the first report on the comprehensive profiling of major chemical components in ZSXBGZD. The results of the study could help to uncover the chemical basis of ZSXBGZD and possess potential value for quality evaluation purpose.

HPLC determination of massoia lactone in fermented Cordyceps sinensis mycelium Cs-4 and its anticancer activity in vitro.

The fermentation product of Cordyceps sinensis mycelium Cs-4 was commonly used as alternative substitutes of natural C. sinensis. Massoia lactone is the dominant component in the volatile oil of Cs-4 mycelium. In this research, we present a high performance liquid chromatography (HPLC) method for the quantitation of massoia lactone in Cs-4 mycelium. The high and stable contents of massoia lactone with values of 2.98-3.77 mg/g, indicated that massoia lactone could be considered as a marker for the quality assessment of this product. The results of MTT and CCK-8 assay showed that Cs-4 mycelium volatiles exhibited cytotoxicity against eight malignant tumor cells (IC50  = 6.0-49.8 µg/ml) in comparison to the anticancer drug 5-fluorouracil (IC50  = 17.0-425.3 µg/ml), and massoia lactone might be the chemical basis for the anticancer effects of Cs-4 mycelium. Compared to the commercial drugs paclitaxel and docetaxel (IC50  = 253-1973 µg/ml), the Cs-4 mycelium volatiles and massoia lactone were discovered to possess inhibitory to taxol-resistant cell lines (IC50  = 1.5-8.6 µg/ml). PRACTICAL APPLICATIONS: Considering that there is still a lack of marker components distinctive to Cs-4 mycelium, the HPLC method represents a useful tool for the quality evaluation of Cs-4 mycelium. Moreover, the volatile oil of Cs-4 mycelium and massoia lactone have prominent anticancer property in vitro. It gives a clue that Cs-4 mycelium, the volatile oil and massoia lactone could be potentially employed in the food and medical industries for its anticancer applications.

Rapid qualitative and quantitative analyses of anthocyanin composition in berries from the Tibetan Plateau with UPLC-quadruple-Orbitrap MS and their antioxidant activities.

Nitraria tangutorum B. (NT), Hippophae rhamnoides L. (HR), Lycium ruthenicum M. (LR), Lycii fructus (LF), Rosa xanthina L. (RX), and Rubuscor chorifolius L. f. (RC) are six berries from Tibetan Plateau. They have been used in traditional folk medicine with a long history, which are rich in anthocyanins. However, detailed study of their anthocyanins remains scarce. Therefore, a method for rapid simultaneous identification and quantification of 12 anthocyanins from berries using UPLC-Quadruple-Orbitrap MS system (UPLC-Q-Orbitrap MS) was established in this work. It was verified with limit of detection (3.86-11.61 µg/L), limit of quantification (3.86-11.61 µg/L), precision (0.95-2.38%), repeatability (0.96-2.08%), stability (0.86-2.31%), mean recovery (95.8-103.1%), recovery range (93.1-107.2%) and RSD less than 5.21%. It was then used in the analysis of anthocyanins in six berries species; 8, 7, 7, 7, 6 and 9 species of anthocyanins have been identified in NT, LF, LR, HR, RC and RX, respectively based on their own retention time and exact mass in positive mode, and for the first time quantified successfully in each berry (31.11 ± 0.42-2978 ± 25.67 µg.g-1). Finally, 2, 2-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging activity (0.92 ± 0.12-5.61 ± 0.23 mM TE/100 g), ferric reducing antioxidant power (FRAP) (1.23 ± 0.15-7.42 ± 0.28 mM TE/100 g) and total antioxidant activity (T-AOC) assays were used to evaluate the antioxidant activities of the six berries.