Inhibitory mechanism of two homoisoflavonoids from Ophiopogon japonicus on tyrosinase activity: insight from spectroscopic analysis and molecular docking.

The inhibition mechanism of two homoisoflavonoids from Ophiopogon japonicus including methylophiopogonanone A (MO-A) and methylophiopogonanone B (MO-B) on tyrosinase (Tyr) was studied by multiple spectroscopic techniques and molecular docking. The results showed that the two homoisoflavonoids both inhibited Tyr activity via a reversible mixed-inhibition, with a half inhibitory concentration (IC50) of (10.87 ± 0.25) × 10-5 and (18.76 ± 0.14) × 10-5 mol L-1, respectively. The fluorescence quenching and secondary structure change of Tyr caused by MO-A and B are mainly driven by hydrophobic interaction and hydrogen bonding. Molecular docking analysis indicated that phenylmalandioxin in MO-A and methoxy in MO-B could coordinate with a Cu ion in the active center of Tyr, and interacted with amino acid Glu322 to form hydrogen bonding, occupying the catalytic center to block the entry of the substrate and consequently inhibit Tyr activity. This study may provide new perspectives on the inhibition mechanism of MO-A and MO-B on Tyr and serve a scientific basis for screening effective Tyr inhibitors.

Interaction between iron and dihydromyricetin extracted from vine tea.

In this research, the interaction between dihydromyricetin (DMY) obtained from vine tea and iron ions (Fe (II) and Fe (III)) was investigated at pH 3.0, 5.0, and 7.0 with UV absorption and fluorescence quenching spectroscopy. The effects of DMY on the stability and solubility of iron ion were also studied. The results showed the presence of iron ions changed the UV absorption spectra of DMY at the experimental pH values. And the fluorescence spectra showed that iron ion had enhanced fluorescence effect on DMY. In addition, DMY was capable of protecting Fe (II) from being oxidized and improving the solubility of Fe (III).

Methylophiopogonanone B of Radix Ophiopogonis protects cells from H2O2­induced apoptosis through the NADPH oxidase pathway in HUVECs.

Methylophiopogonanone B (MO­B), which belongs to a group of homoisoflavonoids, present in Ophiopogon japonicus, has been identified as an active component with antioxidative and anti­tumor properties. The present study investigated whether MO­B may exert protective effects on human umbilical vein endothelial cells (HUVECs) against H2O2­induced injury in vitro, and whether the MO­B effects may be modulated by the NADPH pathway. HUVECs were treated with MO­B in the presence or absence of H2O2. Malondialdehyde (MDA), reactive oxygen species (ROS) levels, and superoxide dismutase (SOD) activity were analyzed to evaluate cell injury and the antioxidative potential of MO­B. The results revealed that MO­B inhibited the production of MDA and ROS, but enhanced SOD activity. Furthermore, MO­B could alleviate H2O2­induced apoptosis in HUVECs, which is consistent with the expression of apoptosis­associated genes and proteins in cells, including Bax/Bcl­2 and caspase­3. To explore the potential mechanism, the present study investigated the effects of MO­B on NADPH­related signaling via the analysis of neutrophil cytochrome b light chain (p22phox) expression, which is the membrane­associated subunit of NADPH oxidase. MO­B could improve the survival of endothelial cells and therefore may be a potential drug in the treatment of cardiovascular diseases.

Application of Ionic Liquid-Based Ultrasonic-Assisted Extraction of Flavonoids from Bamboo Leaves.

Ionic liquids (ILs), known as environmentally benign "green" solvents, were developed as an optimal solvent for the green extraction and separation field. In this paper, an ionic liquid-based ultrasonic-assisted extraction (IL-UAE) of flavonoids (FVs) from bamboo leaves of Phyllostachys heterocycla was developed for the first time. First, 1-butyl-3-methylimidazolium bromide ([Bmim] Br), with the best extraction efficiency, was selected from fifteen ionic liquids with diverse structure, like carbon chains or anions. Then, the influencing parameters of ionic liquid (IL) concentration, liquid-solid ratio, ultrasonic time, and ultrasonic power, were investigated by single factor tests, and further optimized using response surface methodology (RSM). In the optimization experiment, the best conditions were 1.5 mol/L [BMIM]Br aqueous solution, liquid-solid ratio 41 mL/g, ultrasonic time 90 min, and ultrasonic power 300 W. Furthermore, the microstructures of bamboo leaves and the recovery of FVs and [BMIM]Br were also studied. Therefore, this simple, green and effective IL-UAE method has potentiality for the extraction of FVs from bamboo leaves for the large-scale operations.

Molecular cloning and characterization of an ADP-ribosylation factor 6 gene (ptARF6) from Pisolithus tinctorius.

ADP-ribosylation factor 6 (ARF6) is an evolutionarily conserved molecule that has an essential function in intracellular trafficking and organelle structure. To better understand its role during presymbiosis between plant roots and compatible filamentous fungi, the full-length cDNA sequence of ARF6 from Pisolithus tinctorius was cloned and a variety of bioinformatics analyses performed. The full-length sequence was 849 bp long and contained a 549 bp open reading frame encoding a protein of 182 amino acids. A phylogenetic analysis showed that ptARF6 was the ortholog of the ADP ribosylation factor 6/GTPase SAR1 gene from the white-rot basidiomycete Trametes versicolor. A domain architecture analysis of the ARF6 protein revealed a repeat region, which is a common feature of ARF6 in other species. Recombinant ARF6 protein was expressed with an N-terminal 6×His tag and purified using Ni(2+)-NTA affinity chromatography. The molecular mass of the recombinant protein was estimated by SDS-PAGE to be 25 kDa. The recombinant ARF6 protein bound strongly to 18:1 and 18:2 phosphatidic acids. Thus, ARF6 may participate in the signaling pathways involved in membrane phospholipid composition. The intracellular distribution of ptADP6 in HEK239T cells also indicates that ptADP6 may function not only in plasma membrane events but also in endosomal membranes events. Real-time quantitative PCR revealed that the differential expression of ptARF6 was associated with the presymbiotic stage. ptARF6 may be induced by presymbiosis during the regulation of mycorrhizal formation.

Polyprenols from Taxus chinensis var. mairei prevent the development of CCl4-induced liver fibrosis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The aim of this study was to investigate the anti-fibrotic effects and the possible underlying mechanisms of taxus polyprenols (TPs) isolated from the needles of Taxus chinensis var. mairei. MATERIALS AND METHODS: The animals were randomly divided into normal control with vehicles only (olive oil), rat model given CCl4 only, CCl4+low TPs (48 mg/kg), CCl4+medium TPs (120 mg/kg), CCl4+high TPs (300 mg/kg), and CCl4+Polyene phosphatidylcholine (PP, 120 mg/kg). The rat model of liver fibrosis was induced by subcutaneous injection of 40% (v/v) of CCl4 diluted in olive oil (3 mL/kg body weight) twice per week for 8 weeks. Liver histopathological study was performed. Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and albumin (ALB) of the serum were determined for evaluating the liver function. In order to reveal the possible mechanisms of the anti-fibrotic effects, oxidative stress level, hepatic collagen metabolism, and hepatic stellate cells (HSCs) activation were investigated. Furthermore, the mRNA expression of the fibrotic-related factors was measured by the quantitative real-time RT-PCR. RESULTS: TPs successfully attenuated liver injury induced by CCl4 shown by histopathological sections of livers and improved liver function as indicated by decreased ALT, AST and ALP levels and increased ALB levels in serum of the rats. TPs significantly increased the hepatic Cu/Zn SOD and GSH-Px activities along with GSH content while a remarkable decrease in MDA content. Both immunohistochemical staining and mRNA expression levels of α-SMA indicated a profound suppression of HSCs activation. Furthermore, it significantly inhibited the mRNA expression of the pro-fibrotic cytokines Col α1(I), Col α1(Ш), MMP-2, TIMP-1, TIMP-2, PDGF-ß, TGF-ß1, CTGF and TNF-α and restored the hepatoprotective factor HGF. CONCLUSION: These results suggest that the protective effects of TPs in chronic CCl4-induced liver fibrosis might be related with the reduction of oxidative damage, the inhibition of HSCs activation, the down-regulation of pro-fibrogenic stimuli and the protection of hepatocytes.

Polyprenols from the needles of Taxus chinensis var. mairei.

Polyprenols with various pharmacological activities were first isolated from Taxus chinensis var. mairei, which is native to China, and were identified by a high performance liquid chromatography/mass spectrometry (HPLC/MS), (1)H nuclear magnetic resonance ((1)H NMR), (13)C NMR and infrared (IR) spectroscopy. The taxus polyprenols (TPs) had two maxima of polyprenol-17 and polyprenol-21 and different from polyprenols distributed in other plant species. A reversed-phase HPLC method with a simple gradient elution was established, and the proposed combined peak area method for the quantification of polyprenols was verified. The analysis indicated that TPs were present in the old needles at levels as high as 3% and that T. chinensis var. mairei could be an alternative botanical source for the extraction of polyprenols.