Pubescenosides E–K, Seven New Triterpenoid Saponins from the Roots of Ilex pubescens and Their Anti-Inflammatory Activity

Seven new triterpenoid saponins (1–7), together with three known ones (8–10), were isolated from Ilex pubescens. Elucidation of their structures was performed based on high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS), infrared spectra (IR), and nuclear magnetic resonance (NMR) spectroscopic data. The anti-inflammatory activity of the isolates toward lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages was investigated. The results demonstrated that compounds 3, 5, and 6 inhibited the expression of inducible nitric oxide synthase (iNOS) protein in comparison with LPS stimulation in RAW264.7 cells.

Lipopolysaccharide (LPS)-induced macrophages are widely used to study inflammatory responses in vitro [15]. Macrophages are versatile cells that play many roles, and can be activated by external stimuli, such as LPS, to release excessive amounts of inflammatory mediators, including prostaglandin E 2 (PGE 2 ), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) [16,17]. Moreover, iNOS and COX-2 are considered to be the most important inflammatory mediators [16,18]. In our preliminary research, 27 triterpenoid saponins were isolated from Ilex pubescens, and some of them showed remarkable anti-inflammatory activity [19]. However, whether other compounds from this plant have anti-inflammatory activity is still unclear. Therefore, we used an LPS-induced macrophage inflammatory model to determine the effect of 10 compounds on the expression levels of iNOS and COX-2 protein.

Anti-Inflammatory Activity
The anti-inflammatory activity of compounds 1-10 was evaluated by utilizing a LPS-stimulated RAW264.7 cell model. As presented in Figure 4, when compared with LPS stimulation in RAW264.7 cells, compounds 3, 5, and 6 exhibited inhibitory effects on the expression of iNOS protein, and the positive control drug dexamethasone (DEX) notably inhibited the expression of iNOS and COX-2 protein. The effects of DEX intervention suggested that the experimental procedure was adequate.

Discussion
In summary, seven new triterpenoid saponins, named Pubescenosides E-K, together with three known ones, were isolated from the roots of Ilex pubescens. Elucidation of their structures was performed based on extensive spectroscopic analyses. The anti-inflammatory activity of the isolates Figure 4. Effect of compounds 1-10 (IPZ 1-10) on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. RAW264.7 cells were seeded in 24-well plate and cultured overnight. Subsequently, compounds 1-10 and the positive control dexamethasone (DEX) were administered to RAW264.7 cells for 1 h, and LPS (100 ng/mL) was used to stimulate the cells for the final 18 h. Western blotting was performed to analyze protein expression of iNOS, COX-2, and the corresponding values were quantitated by Odyssey v3.0 software. The results are expressed as the mean ± SEM, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. LPS group.

Discussion
In summary, seven new triterpenoid saponins, named Pubescenosides E-K, together with three known ones, were isolated from the roots of Ilex pubescens. Elucidation of their structures was performed based on extensive spectroscopic analyses. The anti-inflammatory activity of the isolates toward lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages was investigated. The results demonstrated that compounds 3, 5, and 6 inhibited iNOS protein expression in LPS-stimulated RAW264.7 cells with dexamethasone as a positive control. The findings revealed that compounds 3, 5, and 6 might have anti-inflammatory activity and might have potential value in anti-inflammatory treatments.

Plant Material
The roots of Ilex pubescens were collected near Conghua City, Guangdong Province, China, in July 2012, and identified by Prof. Guangxiong Zhou of the College of Pharmacy, Jinan University. The voucher specimen (12071002) is stored in the International Institute for Translational Chinese Medicine, Guangzhou University of Traditional Chinese Medicine, Guangzhou, China.