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Background: Hepatocellular carcinoma (HCC), a malignancy with high mortality and recurrence rates, has very limited options for treatment strategies. Dehydrocostus lactone (DL) is the principal quality marker extracted from Aucklandia lappa Decne. and has been demonstrated to possess excellent anticancer activity. The key purpose of this study was to explore the therapeutic effects and potential mechanisms of DL on HCC. Methods: The effect of DL on the cell viability of HCC cell lines (HepG2 and SK-HEP-1) and normal human hepatocyte cell line (L-O2) was examined by CCK8, colony formation, and BeyoClickTMEdU-488 assays; Hoechst 33,258 staining and flow cytometry were utilized to determine the impact of DL on apoptosis; the sensitivity of HCC cells to DL was explored by adding Z-VAD-fmk. In addition, the expression of apoptosis-related proteins Bax, Bcl-2, and PARP was detected by western blotting; the expression of p-H2AX was investigated by western blotting and immunofluorescence staining; cell cycle distribution was observed using flow cytometry; the migration ability and invasiveness of HCC cells were assessed by wound healing assay and transwell assay; immunoblotting was applied to visualize the levels of EMT markers in the two HCC cell lines. Transcriptome sequencing was performed to reveal the underlying mechanisms of DL anti-tumor; western blotting and qRT-PCR were employed to verify the mRNA and protein abundance of p53, p21, and CDK2; HepG2 xenograft in mice was used to test the anti-tumor effects of DL in vivo. Results: DL suppressed the proliferation of HCC cell lines (HepG2 and SK-HEP-1) but had little effect on a normal human liver cell line (L-O2). In addition, DL also induced apoptotic death of the HCC cells by activating Bax and downregulating Bcl-2. The antiproliferative effects of DL could be attributed to increased DNA damage and G1-phase cell cycle arrest. Moreover, DL inhibited HCC cell invasiveness and migration in vitro by decreasing the levels of β-catenin, N-cadherin, and TCF8/ZEB1, and increasing the E-cadherin level. RNA sequencing indicated that DL exerted its anti-hepatoma effects partly via regulation of p53-p21-CDK2 signaling. These results were validated by our in vivo experiments, in which DL markedly suppressed the growth of HepG2 xenografts in a mouse model without any toxic side effects, which corresponded to decreased expression of Ki67 and MMP9 in the tumor tissues. Conclusion: DL has significant anti-cancer effects in both in vivo and in vitro HCC models, and therefore could be further developed as a promising drug for the treatment of HCC.

Narirutin, extracted from the dried ripe pericarp of Rutaceae orange and its cultivated varieties, is a dihydroflavone compound with various biological activities and pharmacological effects. So far as we know, the metabolism profiling of narirutin has been insufficient until now. In the present study, an efficient method was employed to perform rapid analysis and identification of narirutin metabolites by using ultra-high performance liquid chromatography quadrupole exactive orbitrap MS/MS (UHPLC-Q-Exactive Orbitrap MS/MS) combined with an analogous core recursion (ACR) analysis strategy. Firstly, according to the basic core structure of the dihydroflavonoid, the cleavage mode and fragmentation ions of narirutin were summarized. Secondly, based on the difference of the substituent groups, the fragment ions of narirutin were preliminarily inferred and verified by the secondary mass spectra of its standard compound. Thirdly, the fragment ions of narirutin in positive and negative ion modes were summarized to provide a basis for the identification of metabolites. Fourthly, candidate metabolites were accurately and tentatively identified according to the cleavage law of mass spectrometry, literature reports, comparison of reference substances, and especially the diagnostic fragment ion clusters (DFICs) and characteristic suggestive ions (CSIs) deduced preliminarily. Finally, a total of 46 metabolites (30 in vivo and 19 in vitro), including prototype drugs were identified based on the ACR analysis strategy, chromatographic retention behavior of metabolites, corresponding ClogP value and accurate molecular weight. Among them, 22 metabolites were discovered in rats plasma, 12 in urine, 4 in liver tissue, 19 in liver microsomes, respectively. Additionally, metabolites relative contents were also studied by extracted ion chromatography (EIC) method. Our result also illustrated that narirutin primarily underwent glucuronidation, sulfation, glutathione binding, deglycosylation, oxidation, reduction, methylation, hydroxylation, ring-opening and their composite reactions. A novel strategy was constructed to comprehensively elucidate the biotransformation pathways of narirutin in vitro and in vivo, and a systematic metabolic profile of narirutin was generated. This study provided a great reference for further understanding of the biotransformation pathways and guiding for clinical application of narirutin.

Resveratrol is an antitoxin secreted by plants such as Polygonum cuspidatum Sieb. et Zucc and Vitis vinifera L. when they are attacked by pathogens. In the present study, three methods were used to prepare biological samples, and then an efficient strategy based on ultra-high-performance liquid chromatography-linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) was developed to screen and identify resveratrol metabolites in rat urine, plasma and faeces. As a result, a total of 56 resveratrol metabolites were screened and characterized. Among them, 39 metabolites were found in rat urine, while 6 and 16 metabolites were characterized from rat plasma and faeces, respectively. In addition, 56, 12 and 15 metabolic products were screened by solid phase extraction (method I), methanol precipitation (method II) and acetonitrile precipitation (method III), respectively, indicating that method I could be adopted as the most acceptable method. The results also demonstrated that resveratrol mainly underwent glucuronidation, glucosylation, sulfation, hydroxylation, dehydrogenation, hydrogenation, methylation and their composite reactions. Moreover, these metabolic reactions occurred to form a possible metabolic network that is similar to a triangular pyramid model. In summary, this research provides an idea for the further study of drug metabolism.

Magnolol, an essential bioactive natural lignans isolated from Magnolia Officinalis, has various pharmacological activities, such as antibacterial, anti-inflammatory, antioxidation and anti-tumor. In this study, a practical strategy based on ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) was established to reveal the metabolic fate of magnolol in rat plasma and urine. First of all, in the data-dependent scanning (DDS) acquisition mode, the ESI-MSn data sets of biological samples and reference standard were obtained by using a high-quality online data analysis method. Subsequently, off-line data-mining techniques including multiple mass defect filters (MMDFs) and high-resolution extracted ion chromatograms (HREICs) were applied to screen major-to-trace metabolites of magnolol at the full-scan ESI-MS1 level. Finally, a total of 177 metabolites (prototype compound included) were preliminarily observed and characterized according to accurate mass measurement, the characteristic fragmentation patterns, chromato-graphic behaviors, and corresponding Clog P values. The results showed that magnolol experienced a variety of biotransformation reactions in rats, including hydroxylation, methoxylation, dehydrogenation, carboxylation, sulfonation, glucuronide conjugation, glucose conjugation, N-acetylcysteine (NAC) conjugation, glutathione conjugation and their composite reactions. In conclusion, this study not only greatly expanded the understanding of the therapeutic material basis and pharmacological mechanism of magnolol, but also provided new ideas for its toxicity evaluation, safety monitoring and drug delivery forms design.

Studies on herbal medicines and fermentation products have become increasingly essential with the development of modern industry and technology. In order to verify that fermentation can bring about changes, Paecilomyces cicadae [Paecilomyces cicadae (Miquel.) Samson] was used to ferment Astragali radix [Astragalus membranaceus (Fisch.) Bge. var. mongho-licus (Bge.) Hsiao]. After solid fermentation for Astragali radix produced by Paecilomyces cicadae (SF-AP) was established, an efficient strategy based on ultra-high performance liquid chromatography-linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) was developed to screen and identify the chemical transformations in SF-AP and Astragali radix according to the acquired diagnostic product ions (DPIs). As a result, 114 compounds including 45 saponins and 69 flavonoids were finally identified and validated. Moreover, two kinds of antioxidative tests corresponding to the scavenging of DPPH· and ABTS·+ were applied to evaluate the antioxidative activity of Astragali radix before and after fermentation. The results demonstrated that some significant chemical transformations such as relative content fluctuations and structural isomerism owing to the occurrence of hydrolysis and conversion reactions and the antioxidative activity of SF-AP was much higher than that of the Astragali radix. This study could provide a new method for the utilization of Astragali radix and constructive guidance for the further research of fermented herbal medicines.