A New Flavone C-Glycoside from Clematis rehderiana

A new flavone C-glycoside, isovitexin 6″-O-E-p-coumarate (1) and two known flavonoid glycosides—quercetin 3-O-β-D-glucuronopyranoside (2) and isoorientin (3)—were isolated from an ethanol extract of aerial parts of Clematis rehderiana. Their structures were determined by spectroscopic methods. The antioxidant effects of the two flavone C-glycosides were evaluated by both the MTT and DPPH assays. Compound 1 showed potent activities against H2O2-induced impairment in PC12 cells within the concentration range tested, whereas compound 3 scavenged DPPH radical strongly, with an IC50 value of 13.5 μM.


Introduction
The genus Clematis (Ranunculaceae), which comprises about 300 species, is widespread throughout the World. About 147 species (93 endemic ones) are distributed in China, and 56 of these are distributed in Yunnan province [1,2]. It is reported that 77 Clematis species have been used in traditional Chinese medicine, of which 32 are found in Yunnan province [3]. The genus Clematis has many different pharmacological effects such as antibacterial, anti-inflammatory, antitumor, analgesic OPEN ACCESS and diuretic functions [4]. Reports on the chemical components of genus Clematis have been scarce up to now and mainly refer to triterpenoid saponins [4][5][6]. In order to provide some knowledge for better usage of the Clematis resources, we have investigated the chemical constituents and antioxidant activity of Clematis rehderiana from Yunnan.
C. rehderiana (English common name: cowslip scented clematis) is distributed in northwest Yunnan and used by local Tibetan people as a diuretic for eliminating dampness and cure indigestion, lumps in the abdomen and skin ulcers [7]. To the best of our knowledge, there are no reports about the chemical constituents of the species. We report here the isolation and characterization of one new and two known flavonoid glycosides from this plant, as well as antioxidant assay results for two of these compounds.

Structure Elucidation
The extract of C. rehderiana obtained with 90% EtOH was then successively partitioned between H 2 O and EtOAc, followed by H 2 O and n-butanol. The n-butanol soluble fraction was separated by different chromatographic procedures to afford one new C-flavone glycoside 1 and two known flavonoid glycosides 2 and 3. Their structures were determined by examination of their spectral data and comparison of the data with reported literature values.

Biological Activity
Since phenolics are characterized by antioxidant activities [11][12][13][14], two isolates were subjected to antioxidant activity experiments using both the MTT and DPPH assays (Tables 2 and 3). Compound 1 showed potent activity against H 2 O 2 -induced impairment in PC12 cells within the concentration range tested (0.4 to 50 μM), whereas isoorientin (3) scavenged DPPH radical strongly, with an IC 50 value of 13.5 μM. This indicated that compound 1 might be an indirectly acting antioxidant, while 3 might be a directly acting antioxidant.

General
Optical rotations were measured with a JASCO DIP-370 digital polarimeter in MeOH solutions. IR spectra were measured on a Bio-Rad FTS-135 infrared spectrometer with KBr pellets. UV spectra were obtained with a Shimadzu UV-2401PC spectrometer. Mass spectra were measured on a VG Auto Spec-3000 spectrometer. NMR spectra were recorded on a DRX-500 NMR spectrometer with TMS as internal standard. Silica gel (200-300 mesh) for column chromatography and precoated TLC plates (Si gel G) were purchased from the Qingdao Marine Chemical Factory (Qingdao, P. R. China). Reversed-phase C 18 silica gel for column chromatography were obtained from Merck. Sephadex LH-20 for column chromatography was purchased from Amersham Biosciences.

Plant material
Aerial parts of Clematis rehderiana was collected from Lijian in Yunnan Province, China, in August 2005. A voucher specimen (KUN0816301) is stored at the herbarium of Kunming Institute of Botany, Chinese Academy of Sciences.

Antioxidant assays
The antioxidant assay against H 2 O 2 -induced impairment in PC12 cells was conducted according to the reported protocol [14]. Briefly, PC12 cells were seeded into 96-well plates in RPMI 1640 medium with 10% characterized Newborn Bovine Serum. Twenty-four hours later, different concentrations of compounds 1 and 3 together with freshly prepared H 2 O 2 were added and incubation continued for 1 hour. Then MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added and the incubation continued for 4 hours. Finally, solution (100 μL) containing 5% iso-butanol, 10% SDS (Sigma) and 0.004% HCl was added. The mixtures were kept overnight and the index of cell viability (% of control) was calculated by measuring the optical density of the color produced by MTT dye reduction with a microplate reader at 570 nm.
DPPH radical-scavenging activity assays were performed according to our previously reported procedures [11]. Each compound (100 μL) at five different concentrations was added to DPPH solution (100 μL). The absorbance was measured with a microplate reader at 517 nm after 30 min of reaction at 37 °C. IC 50 values denote the concentration of sample required to scavenge 50% DPPH free radicals.

Conclusions
A new flavone C-glycoside, isovitexin 6″-O-E-p-coumarate (1) and two known flavonoid glycosides-quercetin 3-O-β-D-glucuronopyranoside (2) and isoorientin (3)-were isolated from an ethanol extract of aerial parts of C. rehderiana. The antioxidant effects experiments of these compounds by both the MTT and DPPH assays suggested that flavone glycosides were major constituents with antioxidant activities in this plant.