A new flavonol from the kino of Eucalyptus citriodora

Abstract A new flavonol, 6-[1-(p-hydroxyphenyl)ethyl]rhamnocitrin (3) together with two known compounds, kaempferol (4) and 7-O-methyl aromadendrin (5) were isolated from the kino of Eucalyptus citriodora and their structures were elucidated on the basis of spectroscopic methods including 2D NMR spectra. Rhamnocitrin (1), 6-[1-(p-hydroxyphenyl)ethyl]-7-O-methyl aromadendrin (2), previously isolated from this plant and compounds 3−5 were tested for inhibitory activity against 15-lipoxygenase. All compounds exhibited moderate to strong inhibitory activities, of which compounds 2, 3 and 5 showed stronger inhibitory activity (IC50 19.7 ± 0.5, 29.3 ± 0.9 and 31.4 ± 1.0 μM, respectively) than the positive control quercetin (IC50 37.5 ± 0.8 μM).


Introduction
Many species of eucalypts produce kino in response to wounding or disease (Hillis & Hasegawa 1963). Kino generally plays a role in wound defence as a tree resistance mechanism, acting as a barrier zone to protect the tree against pests, diseases and damage (Eyles & Mohammed 2003). Eucalyptus citriodora, a tree native to Australia is widely grown in different parts of the world and is commonly cultivated in Taiwan. This plant rich in essential oil and polyphenols, (Vaghasiya et al. 2008) is traditionally used as analgesic, anti-inflammatory and antipyretic remedies for the symptoms of respiratory infections (Silva et al. 2003). The kino of this plant has been found to be a botanical origin for propolis (Moreno et al. 2000;Freitas et al. 2008). To date, phytochemical studies on E. citriodora kino have described the isolation of triterpenoids, phenolics and flavonoids (Freitas et al. 2007;. Cytotoxic effect on human hepatoma HepG2 Cells (Shen et al. 2012), and antiproliferative activity and apoptosis induction of E. citriodora kino in melanoma B16F10 cells have been investigated . In previous studies, we isolated rhamnocitrin (1) (Tu et al. 2007) and 6-[1-(p-hydroxyphenyl)ethyl]-7-O-methyl aromadendrin (2) ) which exhibited antiatherogenic and antiproliferative activity, respectively.

Results and discussion
The EtOAc fraction was subjected to repeated silica gel column chromatography and further purified by Sephadex LH-20 column chromatography and preparative TLC to afford a new flavonol (3) together with two known compounds (4, 5). Their structures were elucidated on the basis of spectroscopic methods including HR-EI-MS and 2D NMR spectra (COSY, NOESY, HMQC and HMBC) and comparison with the data reported in the literature.

General experimental procedures
Column chromatography (CC) was performed on silica gel 60 (Merck, 70-230 mesh) and Sephadex LH-20 (Pharmacia), respectively. Thin layer chromatography (TLC) and preparative TLC were performed on precoated silica gel plates (Merck, kieselgel 60 F 254 , 0.25 and 1.00 mm). uV spectra were obtained on a Cary 50 uV-visible spectrophotometer. Optical rotations were measured on a JASCO DIP-360 digital polarimeter. IR spectra were taken on a Perkin Elmer 781 infrared spectrophotometer. CD spectra were obtained on a Jasco J-720 spectropolarimeter. NMR spectra were obtained on Bruker AV-500 NMR spectrometer. EI-MS and HR-EI-MS were recorded on a JEOL JMS-700 mass spectrometer. 15-Lipoxygenase from soybean and linoleic acid were purchased from Sigma (St. Louis, MO).

Plant material
Eucalyptus citriodora kino was collected in May 2009 in Yung Kang, Tainan, Taiwan, and identified by Prof. Chang-Sheng Kuoh, Department of Biology, National Cheng Kung university. A voucher specimen (CNuNP0605) was deposited in the natural product laboratory of Department of Medicinal Chemistry, Chia Nan university of Pharmacy and Science.

Measurement of 15-lipoxygenase inhibitory activity
15-Lipoxygenase inhibitory activities were measured according to the method of (Lyckander & Malterud 1992) with minor modification. Borate buffer (200 μL, pH 9.0) was added to 50 μL of different concentrations of compounds 1−5 along with positive control quercetin and enzyme (500 μL, 400 units/mL in borate buffer). The compounds and quercetin were added as DMSO solutions. After incubation of the test solution for 4 min, 250 μL linoleic acid was added and the change in the absorbance was measured for 60 s at 234 nm. The enzyme solution was kept in ice and controls were measured at intervals throughout the experimental periods to ensure that the enzyme activity was constant. Each compound was tested in triplicate and the values are expressed as means ± SD. Quercetin, a well-known inhibitor of 15-lipoxygenase (Lyckander & Malterud 1992) was employed as a positive control.

Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1-S7 and Tables S1-S2.