Enhancement of an In Vivo Anti-Inflammatory Activity of Oleanolic Acid through Glycosylation Occurring Naturally in Stauntonia hexaphylla

Abstract

Stauntonia hexaphylla (Lardizabalaceae) has been used as a traditional herbal medicine in Korea and China for its anti-inflammatory and analgesic properties. As part of a bioprospecting program aimed at the discovery of new bioactive compounds from Korean medicinal plants, a phytochemical study of S. hexaphylla leaves was carried out leading to isolation of two oleanane-type triterpene saponins, 3-O-[β-d-glucopyranosyl (1→2)-α-l-arabinopyranosyl] oleanolic acid-28-O-[β-d-glucopyranosyl (1→6)-β-d-glucopyranosyl] ester (1) and 3-O-α-l-arabinopyranosyl oleanolic acid-28-O-[β-d-glucopyranosyl (1→6)-β-d-glucopyranosyl] ester (2). Their structures were established unambiguously by spectroscopic methods such as one- and two-dimensional nuclear magnetic resonance and infrared spectroscopies, high-resolution electrospray ionization mass spectrometry and chemical reactions. Their anti-inflammatory activities were examined for the first time with an animal model for the macrophage-mediated inflammatory response as well as a cell-based assay using an established macrophage cell line (RAW 264.7) in vitro. Together, it was concluded that the saponin constituents, when they were orally administered, exerted much more potent activities in vivo than their sapogenin core even though both the saponins and the sapogenin molecule inhibited the RAW 264.7 cell activation comparably well in vitro. These results imply that saponins from S. hexaphylla leaves have a definite advantage in the development of oral medications for the control of inflammatory responses.

Keywords: Lardizabalaceae; Stauntonia hexaphylla; anti-inflammatory effect; oleanane triterpene saponin.

Figure 1

The structure of the isolated compounds 1 and 2.

Figure 2

Positive ion mode LC-electrospray ionization (ESI)-MS/MS multiple reaction monitoring (MRM) analysis of EtOH extract of Stauntonia hexaphylla leaves. Profile of the specific transition of compounds 1 and 2 from precursor ion to product ions in the MRM mode.

Figure 3

MS¹ and MS² spectrum of compounds 1 (A) and 2 (B).

Figure 4

Cytotoxic properties of compounds 1 (Comp. 1) and 2 (Comp. 2) RAW 264.7 cells after treatment with compounds 1–2, oleanolic acid (O.A.) and dexamethasone (DEX) (0.08, 0.32, 1.25, 5 and 20 µM, respectively) for 3 days. Dexamethasone was used as the positive control.

Figure 5

Inhibition of the activation of the RAW 264.7 cell line by the active compounds 1 (Comp. 1) and 2 (Comp. 2), in vitro. Cells were treated with different concentrations of compounds 1–2, oleanolic acid (O.A.) and dexamethasone (DEX), separately. Nitrite oxide concentrations in the culture supernatants were determined using a Griess reagent assay. Dexamethasone was used as the positive control.

Figure 6

Inhibition of the activation of macrophage by orally administered compounds in vivo. After 4 days treatment with 20 mg/kg compounds 1 (Comp. 1) and 2 (Comp. 2) and oleanolic acid (O.A.), respectively, LPS was injected i.p., and the blood was drawn for the analysis of the concentration of TNF-α. The concentration of TNF-α was determined with ELISA (* p <0.05, ANOVA). Dexamethasone (Dexa^#) was used as the positive control. Sham means a negative control group that is injected with LPS to induce inflammation but not treated.