Rubescins I and J, further limonoid derivatives from the stem bark of <i>Trichilia rubescens</i> (Meliaceae)

<p>Two new tetranorterpenoid derivatives named rubescins I (<b>1</b>) and J (<b>2</b>), were isolated along with six known compounds including rubescin D (<b>3</b>), lichexanthone (<b>4</b>), scopoletin (<b>5</b>), scopoletin <i>O</i>-glycoside (<b>6</b>), <i>β</i>-sitosterol (<b>7</b>) and stigmasterol (<b>8</b>) from the stem bark of <i>Trichilia rubescens</i> (Meliaceae). The structures of the compounds were determined by means of MS, different NMR and by comparison with related data reported in the literature.</p

Limonoids used in this study.

<p>Limonoids used in this study.</p

TR4 and TR9 induce apoptosis in human hepatoma cells.

<p>HepG2 cells were incubated with 5 μM TR3, TR4 or TR9 for 24 hours. As an apoptosis induction control HepG2 cells were incubated with 80 nM ActD as well as 40 ng/ml TNFα (ActD/TNFα) for 16 hours. Apoptosis was measured by Caspase-3 colorimetric assay (A) or by Western Blot analysis of Caspase-3- and PARP-cleavage (B). HepG2 cells were incubated with 5 μM TR3, TR4 or TR9 for 40 hours or with ActD/TNFα for 16 hours. Apoptotic DNA Ladder was determined by performing DNA-fragmentation analysis (C). HepG2 cells were incubated with 5 μM TR3, TR4, TR9 or with ActD/TNFα for 16 hours (D, E). FACS analysis was performed to detect apoptotic cells stained with propidium iodide (PI) and Annexin V. Representative plots are shown (D). E: Quantification of PI-positive HepG2 cells. F: HepG2 cells were incubated with 5 μM TR3, TR4 or TR9 for 6 hours. Real time RT-PCR for Bcl2 and Bcl10 expression was performed. Relative expression levels were determined using mATPsy expression as a reference. ★★ p ≤ 0.01, ★★★ p ≤ 0.001 vs DMSO controls.</p

TR4 and TR9 inhibit proliferation of human hepatoma cells.

<p>A: HepG2 cells were incubated with 10 μM TR3, TR4 or TR9 for 20 hours. Cell proliferation was measured by BrdU-Assay. DMSO control: 0.1%. B: HepG2 cells were incubated with 5 μM TR3, TR4 or TR9 for 6 hours. Expression of PCNA, p21 and HSA was measured by real time RT-PCR. DMSO control: 0.05%. ★ p ≤ 0.05, ★★★ p ≤ 0.001 vs DMSO controls. C + D: HepG2 cells were incubated with 5 μM TR3, TR4 or TR9 for 24 hours. Protein expression of Cyclin D1 was detected by Western Blot analysis (C) and quantified by using Image Lab^TM Software (D). Quantification was performed in relation to reference expression of GAPDH as a loading control.</p

The limonoids TR4 and TR9 have strong adverse effects on human hepatoma cell viability.

<p>A: HepG2 human hepatoma cells were incubated with 25 μM of 6 different limonoids (TR3, TR4, TR8, TR9, TR11 and TR12, isolated from <i>Trichilia rubescens</i> Oliv.) for 24 hours. Cell viability was measured by MTT assay. DMSO control: 0.25%. B-D: Structures of the most powerful limonoids TR4 and TR9 as well as the structurally similar but ineffective control limonoid TR3. E: HepG2 cells were incubated with 5 μM of TR3, TR4 or TR9 for 6 to 72 hours. Cell viability was measured by MTT assay. The grey line indicates the TC50. DMSO control: 0.05%. F: Primary mouse hepatocytes (PH) isolated from male C57/Bl6 mice, as well as corresponding mouse hepatoma cells (Hepa1-6) were incubated with 10 μM TR3, TR4 or TR9 for 24 hours. Cell viability was measured by MTT assay. DMSO control: 0.1%. ★★ p ≤ 0.01, ★★★ p ≤ 0.001 vs DMSO controls.</p

TR4 and TR9 decrease hepatoma cell viability at a lower TC50 than Sorafenib, SAHA or 5-Fluorouracil.

<p>HepG2 (A, B) or Huh7 (C, D) human hepatoma cells were incubated with 1, 5 or 10 μM TR3, TR4 or TR9 (A, C) or 1, 10 or 100 μM Sorafenib, SAHA or 5-Fluorouracil (5-FU) (B, D) for 24 hours. Cell viability was measured by MTT assay (A-D). Grey lines indicate TC50s. DMSO control: 0.1%. ★★ p ≤ 0.01, ★★★ p ≤ 0.001 vs DMSO controls.</p

qPCR sequences.

<p>qPCR sequences.</p

Oligoamide, a new lactam from the leaves of <i>Angylocalyx oligophyllus</i>

<p>A new lactam, oligoamide (<b>1</b>), along with three known compounds (<b>2</b>–<b>4</b>), stigmasterol-3-O-β-D-glucopyranoside (<b>2</b>), formononetin (<b>3</b>) and (-)-pinitol (<b>4</b>) were isolated from the CH₂Cl₂/CH₃OH (1:1) extract of the leaves of <i>Angylocalyx oligophyllus</i> by chromatographic separation. Their structures were elucidated on the basis of spectroscopic analysis (UV, IR, MS, 1D, and 2D NMR). Compound <b>1</b> was found to have weak antioxidant and urease inhibitory potential.</p