Anti-Inflammatory and Antioxidant Components from Hygroryza aristata

Twenty-six known compounds and two new compounds, including a new lignan, (7S*,8R*,7’R*,8’S*)-icariol A2-9-O-β-xylopyranoside (1), and a new indole alkaloid, hygarine (2), were isolated from the extracts of Hygroryza aristata (Gramineae). The structures of all compounds were elucidated on the basis of NMR spectral analysis. The compounds (-)-epigallocatechin-3-O-gallate (4) and (-)-epicatechin-3-O-gallate (5) possess free radical scavenging activities and compound 1 could inhibit superoxide anion generation and elastase release by fMLP/CB-induced human neutrophils with IC50 values of 19.33 ± 0.86 and 24.14 ± 1.59 μM, respectively.


Introduction
Hygroryza aristata (Gramineae) is a stoloniferous perennial plant widely distributed in tropical Asia [1]. This plant grows in paddy fields and ponds at 400-800 meters above sea level, often forming floating clusters in lakes and slow-flowing rivers, and is a forage favored by cattle. Nevertheless, to our knowledge the chemical and biological activities of this plant have not been reported previously.
In the search for biologically active constituents from natural sources, we determined that the methanolic extract of this plant was able to scavenge DPPH free radicals. In the present study two new compounds, (7S*,8R*,7'R*,8'S*)-icariol A 2 -9-O-β-xylopyranoside (1) and hygarine (2), were isolated together with 26 known compounds from the extracts of the H. aristata (Figure 1). Several of these compounds were evaluated for their antioxidant and anti-inflammatory activities.

General
Melting points were measured on a Yanagimoto MP-S3 micro melting point apparatus and are uncorrected. The UV spectra were recorded on a Hitachi U-3010 spectrophotometer in MeOH solution.
The IR spectra were recorded on a Jasco IR Report-100 spectrophotometer as KBr discs. The 1 H-and 13 C-NMR spectra were recorded on Bruker Avance-400 spectrometer. Chemical shifts are shown in δ values with tetramethylsilane as internal reference. The mass spectra were performed in the EI or FAB (matrix: glycerol) mode on a VG 70-250 S spectrometer. Specific rotations were determined on a Jasco P-1010 polarimeter.

Plant Material
Hygroryza aristata was collected and authenticated by Prof. C. S. Kouh at I-Lang, Taiwan. A voucher specimen (CGU-HA-1) was deposited in the herbarium of Chang Gung University, Taoyuan, Taiwan.

DPPH Free-Radical Scavenging Activity
The scavenging activity of the DPPH radical was assayed by the modified method of Shimada et al. [26]. The absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicates higher free-radical scavenging activity. The DPPH radicalscavengingactivity (%) was calculated by the following equation: scavenging activity (%) = (1 − A sample /A control ) × 100%

Preparation of Human Neutrophils
Human neutrophils from venous blood of healthy, adult volunteers (20-30 years old) were isolated with a standard method of Dextran sedimentation prior to centrifugation in Ficoll Hypaque gradient and hypotonic lysis of erythrocytes. Purified neutrophils that contained > 98% viable cells, as determined by trypan blue exclusion, were resuspended in HBSS buffer at pH 7.4, and kept at 4 °C before use.