Spectrum-Effect Relationship-Based Strategy Combined with Molecular Docking to Explore Bioactive Flavonoids from Sceptridium ternatum.

Sceptridium ternatum is a herbaceous plant with significant potential for pharmaceutical and cosmetic applications. In this study, we established a spectrum-effect relationship-based strategy to investigate the bioactive basis and tissue distribution in S. ternatum. First, a phytochemical analysis on the ethanol extracts from roots, stems, and leaves of S. ternatum was performed using the colorimetric method, high-performance liquid chromatography-ultraviolet (HPLC-UV), and high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS/MS). Then, radical scavenging assays and the lipopolysaccharide-stimulated RAW 264.7 cell model were used to estimate the antioxidant and anti-inflammatory activities, respectively. Spectrum-effect relationship analysis and molecular docking were further employed to evaluate the correlation between the phytochemical profile and anti-inflammatory activity. Our results demonstrate that S. ternatum leaves contained the most abundant flavonoids and exerted the best biological activities. Their IC50 values for scavenging 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) and 1,1-diphenyl-2-picrylhydrazyl radicals were 2.43 ± 0.13 and 5.36 ± 0.54 mg/mL, respectively. In lipopolysaccharide-stimulated RAW 264.7 cells, the leaf extract caused the greatest reduction in nitric oxide production (38.15%) and interleukin-6 release (110.86%). Spectrum-effect relationship analysis and molecular docking indicated that quercetin 3-O-rhamnoside-7-O-glucoside possessed high anti-inflammatory activity by binding with interleukin-6. In conclusion, S. ternatum is a rich source of bioactive flavonoids with potential for exploitation in the prevention and treatment of oxidative stress and inflammation-related pathologies.

Global Metabolomic and Lipidomic Analysis Reveal the Synergistic Effect of Bufalin in Combination with Cinobufagin against HepG2 Cells.

Chansu, which is prepared from the skin secretions of toad (Bufo bufo gargarizans Cantor), is widely used in traditional Chinese medicine (TCM). Being the principal bioactive constituents of Chansu, bufalin (BFL) and cinobufagin (CBF) have been shown to possess anticancer properties. TCM confers bioactivities through the synergistic effect between potential active ingredients, so as to interfere with the development of the disease, and ultimately achieve the therapeutic effect. We found that the anticancer effect was significantly potentiated by cotreatment with BFL and CBF compared to monotreatment, suggesting their synergistic interaction. To reveal their synergistic mechanisms, metabolomic and lipidomic profiling based on liquid chromatography-mass spectrometry (LC-MS) was utilized to delineate the responses in HepG2 cells after treatment with BFL and CBF individually or in combination. Metabolic pathways, including methionine metabolism, energy metabolism, lipid metabolism, and amino acid metabolism, were modulated and subsequently led to apoptosis and cell cycle arrest of HepG2 cells. In particular, the discrepant regulation of methionine metabolism between the monotreatment and cotreatment with BFL and CBF may account for their synergistic effect. Our study provided novel insights into the mechanistic links between cellular metabolism and the synergistic effect, which may ultimately lead to better treatments for hepatoma.

Systems Pharmacology-Based Approach to Comparatively Study the Independent and Synergistic Mechanisms of Danhong Injection and Naoxintong Capsule in Ischemic Stroke Treatment.

To provide evidence for the better clinical use of traditional Chinese medicine preparations (TCMPs), comparison of the pharmacological mechanisms between TCMPs with similar therapeutic effect is necessary. However, methodology for dealing with this issue is still scarce. Danhong injection (DHI) and Naoxintong capsule (NXT) are representative TCMPs for ischemic stroke (IS) treatment, which are also frequently used in combination. Here they were employed as research objects to demonstrate the feasibility of systems pharmacology approach in elucidation of the independent and combined effect of TCMPs. By incorporating chemical screening, target prediction, and network construction, a feasible systems pharmacology model has been established to systematically uncover the underlying action mechanisms of DHI, NXT, or their pair in IS treatment. Systematic analysis of the created TCMP-Compound-Target-Disease network revealed that DHI and NXT shared common targets such as PTGS2, F2, ADRB1, IL6, ALDH2, and CCL2, which were involved in the vasomotor system regulation, blood-brain barrier disruption, redox imbalance, neurotrophin activity, and brain inflammation. In comparative mechanism study, the merged DHI/NXT-IS PPI network and pathway enrichment analysis indicated that DHI and NXT exerted the therapeutic effects mainly through immune system and VEGF signaling pathways. Meanwhile, they had their own unique pathways, e.g., calcium signaling pathway for DHI and gap junction for NXT. While for their synergistic mechanism, DHI and NXT participated in chemokine signaling pathway, T cell receptor signaling pathway, VEGF signaling pathway, gap junction, and so on. Our study provided an optimized strategy for dissecting the different and combined effect of TCMPs with similar actions.

Investigation of the reverse effect of Danhong injection on doxorubicin-induced cardiotoxicity in H9c2 cells: Insight by LC-MS based non-targeted metabolomic analysis.

Although Danhong injection (DHI) has been clearly shown to attenuate ischemic myocardial injury and improve heart function, there is no research regarding its role in doxorubicin (DOX)-induced cardiomyopathy. In this study, we aimed to investigate the reverse effect of DHI on DOX-induced cardiotoxicity in H9c2 cells. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay demonstrated that DHI had no cytotoxicity towards the relevant cell line unless the concentration was as high as 50 µL/mL. The satisfactory cardioprotective effect of DHI exerted at the concentration of 10 µL/mL, which agreed well with the result of real-time cell viability assay. Then non-targeted metabolomics based on LC-MS was employed to characterize metabolic alterations in DOX-induced cells with DHI treatment. Multivariate analysis, including PCA and PLS-DA, revealed 31 altered metabolites after DOX treatment that were primarily related to the disturbance of amino acids and nucleotides metabolism. While DHI could intervene in some disturbed metabolic pathways, such as the metabolism of arginine, glutathione (GSH), pantothenic acid, cytidine, inosine and 5'-methylthioadenosine. These results suggested that DHI exerted the therapeutic effect by improving energy metabolism and attenuating oxidative stress. The present study can lay a foundation for further research on the promising therapeutic effect of DHI in managing DOX-induced cardiotoxicity.

Rapid screening of brain-penetrable antioxidants from natural products by blood-brain barrier specific permeability assay combined with DPPH recognition.

Screening brain-penetrable antioxidants from natural products is a promising way for neuroprotective drug discovery. However, there is no screening methodology enables simultaneous investigation of antioxidant activity and blood-brain barrier (BBB) permeability of compounds from complex samples. Here we propose a novel strategy by combining BBB specific parallel artificial membrane permeability assay with 1,1-diphenyl-2-picrylhydrazyl recognition (BBB-PAMPA-DPPH) to achieve rapid multicomponent screening. First, BBB specific artificial membrane was constructed to separate the compounds with high BBB permeability in herbal extracts. The antioxidant activity of the isolated compounds could be optically recognized through the bleaching of the purple-colored DPPH. By off-line combined HPLC-UV/Q-TOF-MS analysis, the exact BBB-penetrable compounds responsible for the antioxidant activity could be rapidly screened. With this approach, compound 2,6,4'-trihydroxy-4-methoxybenzophenon in Rhizoma Anemarrhena was found to be an antioxidant with very high BBB permeability, which could also be detected in rat plasma and brain tissue after oral administration. Our findings suggested the BBB-PAMPA-DPPH method could be a powerful tool for neuroprotective drug discovery from natural products.

Chemical profiling and antioxidant evaluation of Yangxinshi Tablet by HPLC-ESI-Q-TOF-MS/MS combined with DPPH assay.

Yangxinshi Tablet (YXST) is a Chinese patent medicine commonly used to treat cardiovascular diseases. However, its detailed chemical basis and mechanisms of action have not been clarified. In this study, high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS) was applied for comprehensive analysis of the chemical constituents in YXST. A total of 127 compounds, including 19 phenolic acids, 12 alkaloids, 51 flavanoids, 32 triterpenoids, 2 lignans, 2 phenylethanoid glycosides, 2 anthraquinones, 1 coumarin, and 6 other compounds, were identified or tentatively deduced by comparing their retention times and MS spectra with those of authentic standards or literature data. To further prove the antioxidant activity of YXST, its free radical scavenging capacity was assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) spectrophotometric assay and the antioxidants in YXST were rapidly screened by DPPH-HPLC experiment. Especially, salvianolic acid A and salvianolic acid B showed excellent DPPH scavenging activities with the IC50 of 151.9 and 275.6µg/mL, respectively, which were stronger than that of l-ascorbic acid (positive control) with the IC50 of 297.1µg/mL. Additionally, these two most potent antioxidants were detectable in rat plasma after oral administration. In conclusion, this study reported important clues for the further pharmacological and clinical studies of YXST. Meanwhile, it provided a practical strategy for rapid screening and identifying of in vivo antioxidant in traditional Chinese medicine preparations.

Drug-protein binding of Danhong injection and the potential influence of drug combination with aspirin: Insight by ultrafiltration LC-MS and molecular modeling.

Danhong injection (DHI) is a widely used Chinese medicine injection (CMI) for the clinical treatment of cardiovascular and cerebrovascular diseases. In this study, a simple and efficient in vitro method based on ultrafiltration LC-MS and molecular modeling has been developed to study the human serum albumin (HSA) binding of the compounds in DHI. Seven major components including protocatechuic aldehyde, p-coumaric acid, salvianolic acid D, rosmarinic acid, salvianolic acid E, lithospermic acid and salvianolic acid B were identified as HSA ligands and their binding degrees in the proposed non-saturated model were 26.17, 37.69, 99.77, 91.78, 96.91, 99.42 and 98.10%, respectively. Considering the drug-HSA binding property of the compounds in DHI may change during drug combination therapy, competitive binding assay was carried out to evaluate the influence of aspirin on the DHI-HSA binding. Experimental results revealed that the salvianolic acids in DHI had stronger binding ability to HSA than sodium salicylate. To further verify the results above, molecular modeling and probe displacement assay were conducted to investigate the optimum binding site and binding affinity of the ligands on HSA. Our findings suggested that the established method could be a powerful tool to study the drug-HSA binding property of CMIs.