Three New Dimers and Two Monomers of Phenolic Amides from the Fruits of Lycium barbarum and Their Antioxidant Activities

The aims of this study were to complement the current knowledge on the antioxidative composition of alcohol extracts from the fruits of Lycium barbarum and to evaluate their antioxidant activities. Three new dimers of phenolic amides, named lyciumamides A (<b>3</b>), B (<b>4</b>), and C (<b>5</b>), together with two monomers, <i>N</i>-<i>E</i>-coumaroyl tyramine (<b>1)</b> and <i>N</i>-<i>E</i>-feruloyl tyramine (<b>2</b>), were isolated from the fruits for the first time with the help of activity-guided chromatography. Compounds <b>1</b>–<b>5</b> were evaluated for their antioxidant activities in scavenging 2,2-diphenyl-1-picrylhydrazyl free radical and inhibiting lipid peroxidation in rat liver microsomes induced by ascorbate/Fe²⁺, cumine hydroperoxide, or CCl₄/reduced form of nicotinamide-adenine dinucleotide phosphate, and the results showed that all of them exhibited strong activities, whereas compounds <b>1</b> and <b>2</b> were more potent than the reference <i>tert</i>-butyl-4-hydroxyanisole

MTT assay.

<p>Saponin 1 significantly inhibited the cell viabilities of glioblastoma U87MG and U251MG cells in a dose concentration- and time-dependent manner, but did not affect the cell viability of primary cultured astrocytes, as compared to the vehicle-controls. </p

Saponin 1 Induces Apoptosis and Suppresses NF-κB-Mediated Survival Signaling in Glioblastoma Multiforme (GBM)

<div><p>Saponin 1 is a triterpeniod saponin extracted from <i>Anemone taipaiensis</i>, a traditional Chinese medicine against rheumatism and phlebitis. It has also been shown to exhibit significant anti-tumor activity against human leukemia (HL-60 cells) and human hepatocellular carcinoma (Hep-G2 cells). Herein we investigated the effect of saponin 1 in human glioblastoma multiforme (GBM) U251MG and U87MG cells. Saponin 1 induced significant growth inhibition in both glioblastoma cell lines, with a 50% inhibitory concentration at 24 h of 7.4 µg/ml in U251MG cells and 8.6 µg/ml in U87MG cells, respectively. Nuclear fluorescent staining and electron microscopy showed that saponin 1 caused characteristic apoptotic morphological changes in the GBM cell lines. Saponin 1-induced apoptosis was also verified by DNA ladder electrophoresis and flow cytometry. Additionally, immunocytochemistry and western blotting analyses revealed a time-dependent decrease in the expression and nuclear location of NF-κB following saponin 1 treatment. Western blotting data indicated a significant decreased expression of inhibitors of apoptosis (IAP) family members,(e.g., survivin and XIAP) by saponin 1. Moreover, saponin 1 caused a decrease in the Bcl-2/Bax ratio and initiated apoptosis by activating caspase-9 and caspase-3 in the GBM cell lines. These findings indicate that saponin 1 inhibits cell growth of GBM cells at least partially by inducing apoptosis and inhibiting survival signaling mediated by NF-κB. In addition, <i>in vivo</i> study also demonstrated an obvious inhibition of saponin 1 treatment on the tumor growth of U251MG and U87MG cells-produced xenograft tumors in nude mice. Given the minimal toxicities of saponin 1 in non-neoplastic astrocytes, our results suggest that saponin 1 exhibits significant <i>in vitro</i> and <i>in vivo</i> anti-tumor efficacy and merits further investigation as a potential therapeutic agent for GBM.</p> </div

Saponin 1 treatment resulted in significant apoptotic morphological changes in glioblastoma U87MG and U251MG cells.

<p>A, inverted microscopic observation. B, Nuclear fluorescent Hoechst 33342 staining. C, electron microscopic observation. D, electrophoresis of cellular DNA, lane 0, marker; lane 1-3, primary cultured astrocytes exposed to 7.4 μg/mL saponin-1 for 0, 24, and 72 hours; lane 4-6, U87MG cells exposed to 7.4 μg/mL saponin-1 for 0, 24, and 72 hours; lane 7-9, U251MG cells exposed to 7.4 μg/mL saponin-1 for 0, 24, and 72 hours.</p

Western blotting analysis.

<p>Saponin 1 treatment resulted in a time-dependent decrease in the expressions of NF-κB p65, survivin and XIAP in glioblastoma cell lines. The ratio of Bcl-2/Bax rapidly diminished and the activation of caspase-9 and caspase-3 was observed. Endogenous expression of these proteins was not changed in primary cultured astrocytes exposed to saponin-1-treatment.</p

Nuclear NF-κB p65 expression.

<p>A. Nuclear expression of NF-κB p65 was obviously down-regulated in a time-dependent manner in both U87MG and U251MG cells. In contrast, it was not affected in primarily cultured astrocytes. B. statistical histogram.</p

Chemical structure and HPLC analysis of saponin 1.

<p>A and B: HPLC with different solvent conditions was carried out to establish the purity of saponin 1 on a Dionex P680 liquid chromatograph equipped with a UV170 UV/Vis detector using a YMC-Pack R&D ODS-A column (20×250 mm, YMC Co., Ltd). C: Chemical structure of saponin 1.</p

<i>In vivo</i> anti-tumor effect of saponin 1 in nude mice.

<p>Saponin 1 significantly inhibited tumor growth of U87MG and U251MG cells-produced xenograft tumors. A and B. Typical features of tumoral nodules in nude mice. C. Increase in tumor volume was makedly inhibited in saponin 1-treated nude mice. D. Tumor weight was markedly down-regulated in saponin 1-treated nude mice.</p

NF-κB p65-specific immunocytochemistry in glioblastoma U87MG and U251MG cells.

<p>A, representative immunocytochemical photos targeting NF-κB p65 in primary cultured astrocytes and glioblastoma cells. B, IRS scoring of intracellular expression of NF-κB p65. C, IRS scoring of nuclear NF-κB p65. </p

Structure Guided Discovery of Novel Pan Metallo-β-Lactamase Inhibitors with Improved Gram-Negative Bacterial Cell Penetration

The use of β-lactam (BL) and β-lactamase inhibitor combination to overcome BL antibiotic resistance has been validated through clinically approved drug products. However, unmet medical needs still exist for the treatment of infections caused by Gram-negative (GN) bacteria expressing metallo-β-lactamases. Previously, we reported our effort to discover pan inhibitors of three main families in this class: IMP, VIM, and NDM. Herein, we describe our work to improve the GN coverage spectrum in combination with imipenem and relebactam. This was achieved through structure- and property-based optimization to tackle the GN cell penetration and efflux challenges. A significant discovery was made that inhibition of both VIM alleles, VIM-1 and VIM-2, is essential for broad GN coverage, especially against VIM-producing P. aeruginosa. In addition, pharmacokinetics and nonclinical safety profiles were investigated for select compounds. Key findings from this drug discovery campaign laid the foundation for further lead optimization toward identification of preclinical candidates