Triterpenoids from the Roots of Rhaphiolepis indica var. tashiroi and Their Anti-Inflammatory Activity

Two new triterpenoids, 2α,3β-dihydroxyolean-11,13(18)-dien-19β,28-olide (1) and 3β,5β-dihydroxyglutinol (2), together with eight known compounds (3–10) were isolated from the roots of Rhaphiolepis indica var. tashiroi (Rosaceae). The structures of 1–10 were determined by spectroscopic techniques. Among these isolates, 2α,3β-dihydroxyolean-13(18)-en-28-oic acid (9) exhibited inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide production, with an IC50 value of 16.50 μM.


General Experimental Procedures
All melting points were measured on a Yanaco micro-melting apparatus and were uncorrected. Optical rotations were measured on a Jasco P-1020 digital polarimeter. The UV spectra were obtained with a Jasco V-530 UV/VIS spectrophotometer, and IR spectra (KBr or neat) were taken on a Perkin-Elmer System 2000 FT-IR spectrometer. 1D ( 1 H, 13

Plant Material
The dried roots of R. indica var. tashiroi were collected at Wutai, Pingtung County, Taiwan

Evaluation of O 2 •− Release by Human Neutrophils
The anti-inflammatory effects of the compounds isolated from the roots of R. indica var. tashiroi were evaluated by measuring the inhibition of superoxide anion production. Superoxide anion production was tested using continuous spectrophotometric analysis of ferricytochrome c reduction by an isolated preparation of human neutrophils.

Preparation of Human Neutrophils
Human neutrophils from the venous blood of healthy [11], adult volunteers (20-28 years old) were isolated using a standard method of dextran sedimentation, before centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of the erythrocytes [12]. The purified neutrophils containing >98% viable cells, as determined by the Trypan blue exclusion method, were resuspended in a Ca 2+

Statistical Analysis
The results are expressed as the means ± SEM, and comparisons were made with the Student's t test. A probability of 0.05 or less was considered significant.

Conclusions
There are about five Rhaphiolepis species in India, southeastern and eastern Asia and one species with three varieties in Taiwan [1]. The leaves of R. umbellata were previously reported to have dibenzofurans [14], biphenyls [14][15][16] as phytoalexin with antifungal activity, and its bark contained flavanol glycosides [17] and procyanidins [18]. Two new triterpenoids were isolated from the roots of R. indica var. tashiroi. To our knowledge, this is the first report of triterpenoids from the Rhaphiolepis plants. The inhibition of superoxide production of the Rhaphiolepis genus has never been examined by other research groups. However, we previously reported anti-inflammatory biphenyls and dibenzofurans from the roots of this Formosan plant [2]. Compound 9 was previously reported to show the anti-inflammatory activity [4]. In this study, 9 showed stronger inhibition of superoxide production (IC 50 16.50 ± 0.56 μM) than ibuprofen (IC 50 27.53 ± 3.58 μM) suggesting the possiblity of developing a new anti-inflammatory agent. This result implied that the anti-inflammatory mechanism of action is worth studying further.