Polyphenol-enriched Penthorum chinense Pursh ameliorates alcohol-related liver injury through Ras/Raf/MEK/ERK pathway: Integrating network pharmacology and experiment validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Penthorum chinense Pursh (PCP) has acknowledged as an edible herbal medicinal plant for the prevention and treatment of alcoholic liver injury (ALI). However, only few of researches focus on the chemical material basis and potential mechanisms of PCP against ALI. AIM OF THE STUDY: Herein, we explored the therapeutic effects of PCP extract against ALI based on the integration of network pharmacology, molecular docking, and experiment validation. METHODS: Based on the standard quality control of PCP herbs by UPLC fingerprint and quantitative determination, 80% ethanol extract fraction of PCP containing more polyphenols, compared to aqueous extract fraction of PCP, were chosen for further experiments. After oral administration of PCP ethanol extract, serum pharmacochemistry based on UPLC-Q-Exactive-MS analysis was implemented to evaluate the potential effective compounds. These absorbed prototypes in PCP were used to construct network pharmacology and predict the potential mechanisms of PCP extract against ALI. Then, the predicted targets and biological mechanisms of PCP extract were validated using animal experiments and molecular docking analysis. RESULTS: Although totally 19 polyphenol compounds were identified in PCP ethanol extract by UPLC-MS analysis, only 18 absorbed prototypes were found in the serum collected from mice at 1 h post-administration with PCP extract. These candidate active compounds were further screened into 13 compounds to construct network pharmacology and 433 targets were identified as PCP targets. GO and KEGG pathway enrichment analyses indicated that the effects of PCP extract would involve in Ras signaling pathway. The animal experiments on chronic ALI model mice shown that the oral administration of PCP can alleviate ALI by attenuating hepatic oxidative stress, inflammation and down-regulating the target proteins in Ras/Raf/MEK/ERK pathway. Molecular docking analysis revealed the good binding ability between the three polyphenols (i.e. quercetin, apigenin, thonningianin B) in PCP with the top contribution in network pharmacology, and these target proteins (Ras, Raf, MEK1/2, and ERK1/2). CONCLUSION: Our results clarified that PCP ethanol extract could effectively alleviate ALI by down-regulating Ras/Raf/MEK/ERK signaling pathway promisingly. Quercetin, apigenin, and thonningianin B may be the active compounds of PCP, attributing to the intervention benefits of PCP against ALI.

Identification of neolignans with PCSK9 downregulatory and LDLR upregulatory activities from Penthorum chinense and the potential in cholesterol uptake by transcriptional regulation of LDLR via SREBP2.

ETHNOPHARMACOLOGICAL RELEVANCE: Penthorum chinense has been used in East Asia for the treatment of cholecystitis, infectious hepatitis, jaundice and to treat liver problems. Recent evidences provided the potential for the clinical use of P. chinense in the treatment of metabolic disease. AIM OF THE STUDY: Based on the traditional use and recent evidences, we investigated the effects of constituents from P. chinense with modulation on proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR) expression, and the effect of the most active substance on cholesterol uptake, and genes relevant to lipid metabolism. MATERIALS AND METHODS: The isolation of compounds from the BuOH-soluble extract of 80% methanol extract of P. chinense was conducted using chromatographic methods and the structures were established by interpreting spectroscopic data. Quantitative real time-PCR, and Western blot analysis were performed to monitor the regulatory activity on PCSK9 and LDLR expression. PCSK9-LDLR binding interaction was also tested. The cholesterol uptake in hepatocyte was measured using 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI)-labeled LDL cholesterol. Additionally, gene network analysis of LDLR and responses of its target proteins were carried out to discover genes germane to the effect of active compound on HepG2 cells. Moreover, we performed protein-protein interaction analysis via String and constructed the compound target network using Cytoscape. RESULTS: Two new neolignans and 37 known compounds were characterized from P. chinense. Of the isolated compounds, (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3), penthorin A (4) and methyl gallate (25) were found to suppress PCSK9 mRNA expression with IC50 values of 5.13, 15.56 and 11.66 µM, respectively. However, all the isolated compounds were found to be inactive in PCSK9-LDLR interaction assay. Additionally, a dibenzoxepine-type lignan analog, (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3) demonstrated to upregulate LDLR mRNA and protein expression via transcriptional factor sterol regulatory element-binding protein 2 (SREBP2). Furthermore, (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3) increase the LDL-cholesterol uptake in DiI-LDL assay. CONCLUSION: (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3) seemed to increase potentially cholesterol uptake via the downregulation of PCSK9 and the activation of LDLR in hepatocytes. Moreover, SREBP2 was found to play an important role in regulation of PCSK9 and LDLR by (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one.

Characterization of the anti-Staphylococcus aureus fraction from Penthorum chinense Pursh stems.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) causes serious infections in hospitals. Penthorum chinense Pursh (PCP), employed by the Miao ethnic minority in China, presents antibacterial activities. In this study, the anti-Staphylococcus aureus activities in the pinocembrin-7-O residue-rich fraction from PCP (PGF) were evaluated and characterized. METHODS: The PGF was prepared with 70% ethanol reflux extraction followed by fractional extraction and column chromatography. Pinocembrin-7-O residue components were identified with electrospray ionization mass spectrometry (ESI-MS). Anti-S. aureus activities of the fraction and the main components were evaluated in vitro with serially diluted microbroth assays. Cytotoxicity was evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) chromogenic assays using the NCTC 1469 cell line. RESULTS: This study indicated that the PGF and three components (S1, S2, and S3) presented anti-S. aureus activities, including against clinically isolated MRSA strains. The molecular masses of S1, S2, and S3 were identical to those of pinocembrin-7-O-[4″,6″-hexahydroxydiphenoyl (HHDP)]-ß-D-glucose, pinocembrin-7-O-[3″-O-galloyl-4″,6″-(s)-HHDP]-ß-D-glucose, and Thonningianin A, respectively. The PGF, S1, S2, and S3 all presented an identical minimum inhibitory concentration (MIC) against S. aureus ATCC 25923 and ATCC 43300, which was 62.5 µg/mL. The minimum bactericidal concentrations (MBCs) of the PGF and S3 against ATCC 25923 were 125 and 250 µg/mL, and the MBCs of the PGF, S2, and S3 against ATCC 43300 were 250, 500, and 250 µg/mL, respectively. A time-kill assay consistently indicated that none of the bacterial clones of ATCC 25923 and ATCC 43300 could survive under 2× and 4× MIC PGF treatment for 24 h, respectively. In contrast, 104 CFU (colony-forming units) of ATCC 25923 and ATCC 43300 were killed by 8× and 4× MIC S3 within 24 h, respectively. Additionally, 1×, 2×, and 4× MIC the PGF presented similar postantibiotic effects (PAEs) on the strain ATCC 25923. However, the PAE of the PGF on the strain ATCC 43300 was concentration dependent (1× < 2× < 4× MIC). Finally, the PGF (200 µg/mL) and S3 (60 µg/mL) showed no cytotoxicity against human hepatoma cells. CONCLUSIONS: The PGF and S3 from PCP present potential for the treatment of S. aureus and MRSA infections. The components S1 and S2 present inhibition activities against S. aureus.

Functional Teas from the Stems of Penthorum chinense Pursh.: Phenolic Constituents, Antioxidant and Hepatoprotective Activity.

Penthorum chinense Pursh (PCP), a medicinal and edible plant, is traditionally used for liver protection and treatment of liver diseases. In this study, we compared the differences of composition and activity of flowers, stems and leaves of PCP to select a bioactive part. The stems of PCP with stronger antioxidant activity (6.25-100 µg/mL) and lower cytotoxicity (25-200 µg/mL) than the flowers and leaves were a better bioactive part. Then the chemical composition and hepatoprotective effects of an aqueous extract and an 70% ethanolic extract made with stems of PCP were investigated. We found that the 70% ethanolic extract enriched more polyphenols and flavonoids and possessed significantly stronger hepatoprotective activity than the aqueous extract in the dose range of 25-200 µg/mL, which indicated that 70% ethanol is the better solvent of PCP in extraction technology. Moreover, ethyl acetate extract of stems of PCP (PSE) was used to evaluate the hepatoprotective ability of PCP against oxidative damage using an in vitro model of a normal rat's liver cell (BRL-3A). Besides, 12 phenolic compounds were identified from PSE by ultra-performance liquid chromatography followed by electrospray ionization mass spectrometry (UPLC-ESI-MS). Obtained results strongly support the traditional use of PCP and prove stems of PCP to be an important source of bioactive compounds associated with hepatoprotective activity.

Behavioral responses of gravid Culex quinquefasciatus, Aedes aegypti, and Anopheles quadrimaculatus mosquitoes to aquatic macrophyte volatiles.

Mosquitoes use many cues to assess whether a habitat is conducive for reproduction, possibly including the presence of stimuli from aquatic macrophytes. The effect of water infusions of water hyacinth (Eichhornia crassipes), water lettuce (Pista stratioles), parrotfeather (Myriophyllum aquaticum), and water pennywort (Hydrocotyle umbellata) on mosquito oviposition and attraction was investigated. Gravid Culex quinquefasciatus deposited significantly more egg rafts in water hyacinth, water lettuce, or Bermuda hay (positive control) infusions compared to water, while water pennywort and parrotfeather infusions did not differ from water. In-flight attraction responses of Cx. quinquefasciatus, Aedes aegypti, and Anopheles quadrimaculatus were evaluated. The strongest attraction of gravid Cx. quinquefasciatus and Ae. aegypti occurred in the presence of volatiles from infusions of water hyacinth and water lettuce, which were equal in attractiveness to hay infusion. Water pennywort and parrotfeather infusions were not attractive. Gravid An. quadrimaculatus were not attracted to aquatic plant volatiles. The results suggest that water hyacinth and water lettuce emit volatile chemicals that attract two of three mosquito species tested and stimulate oviposition by Cx. quinquefasciatus, demonstrating that the level of attraction of aquatic plant volatiles varies among species in ways that may have relevance to bait-based detection and control methods.