A new flavonol glycoside from the aerial parts of Epimedium koreanum Nakai

Abstract A new prenylated flavonol glycoside (1) was isolated from a 95% methanol extract of the dried and powdered aerial parts of Epimedium koreanum Nakai (Herba Epimedii), along with seven previously known flavonoids (2–8). The chemical structure of the new compound (1) was established to be 5-hydroxy-4′-methoxy-8-(2-hydroxy-3-methyl-3-butenyl)flavone 3-O-α-l-rhamnopyranosyl-7-O-β-D-gluco pyranoside on the basis of spectroscopic methods. The antioxidant activities of these compounds were determined by the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay and kaempferitrin (8) showed a high reactivity with DPPH.


Introduction
Herba Epimedii (Yinyanghuo in Chinese), the dried aerial parts of various Epimedium plants, has been safely used as an important traditional Chinese herbal medicine for thousands of years (Guo & Xiao 2003). Plants of the genus Epimedium are geographically distributed in China, Korea, Japan, North Italy and some countries in South-east Asia (Fan & Quan 2012). Herba Epimedii, mainly used as a kidney tonic, aphrodisiac and anti-rheumatic medicinal herb (Zhao et al. 2007), has also been used to treat many diseases such as cardiovascular diseases, osteoporosis, sexual dysfunction and tumours (Sze et al. 2010;Ma et al. 2011). Previous studies on the chemical constituents of the Epimedium species have led to the isolation of various flavones, lignans, diphydrophenanthrenes, ionones, terpene glycosides and phenylethanoids (Matsushita et al. 1991;Sun et al. 1998;Li et al. 2011;Zhou et al. 2015). These studies have shown that the Epimedium species are a good source of flavonol glycosides, which exhibit antioxidant, anti-osteoporosis, androgenic and antidepressant-like actions (Pan et al. 2007;Zhang et al. 2008;Li et al. 2012;Miura et al. 2015). In the course of our search for potent bioactive components from Epimedium species, the chemical investigation of the aerial parts of E. koreanum Nakai was carried out. This paper describes the isolation and structure elucidation of one new flavonol glycoside (1) along with seven known flavonoids (2-8). In addition, the antioxidant activities of these compounds were determined using DPPH radical scavenging assay.
The connectivities of 2″-hydroxy-3″-en-anhydroicaritin and glucose and rhamnose were elucidated by the HMBC correlation signals. The HMBC correlations between C-7 (δ C 161. DPPH radical scavenging activities of the flavonoids were determined by spectrophotometric assay. However, only the non-prenylated compound 8 showed high reactivity with DPPH (IC 50 45.5 μg/mL), which was a little weaker than the positive control Vitamin C (IC 50 36.9 μg/mL) (Table S1). Therefore, it can be indicated that C-8 prenylation could reduce the antioxidant activities of the flavonol glycosides isolated from E. koreanum, and this notion was the same as the result previously reported (Li et al. 2012).

General experimental procedures
uV spectra were recorded on a JASCo V-530 spectrophotometer in MeoH, and Ir spectra were obtained on a JASCo FT/Ir-300E spectrometer in KBr discs. one-dimensional (1D) NMr experiments such as 1 H (500 MHz) and 13 C (125 MHz) NMr experiments, 2D NMr experiments such as homonuclear correlation spectroscopy (CoSY), heteronuclear single quantum correlation (HSQC) and HMBC spectra were measured on a Varian unity Inova-500 spectrometer with tetramethylsilane (TMS) as internal standard. The chemical shift values are reported in ppm units, and the coupling constants are in Hz. Hr-ESI-MS data were recorded on a Synapt G2 mass spectrometer (Waters, uK) at the Korea Basic Science Institute. TLC analyses were carried out using pre-coated silica gel 60 F 254 plates (Merck, Darmstadt, Germany) with CHCl 3 /MeoH (8 : 2, volume ratio) and visualisation of the TLC plates was performed under uV radiation (254 nm) and spraying with anisaldehyde-H 2 So 4 followed by heating. Column chromatographic isolations were performed using silica gel 60 (70-230 mesh, Merck). The semi-preparative HPLC system consisted of a chromatographic pump (Waters 600E Multi Solvent Delivery System) and a uV detector (Waters 996 PDA). A Phenomenex Luna C 18 column (10 × 250 mm, particle size 5 μm) was used for separations.

Plant material
The aerial parts of E. koreanum Nakai were purchased from Kyungdong oriental Herbal Market in Seoul, Korea, August 2014 and identified by one of the authors (ISL). A voucher specimen (CNu-HE-201408) has been deposited in the College of Pharmacy, Chonnam National university.

Acid hydrolysis of compound 1
Compound 1 (2 mg) was heated in 10% HCl (3 mL) at 80 °C for 2 h. The reaction mixture was then extracted with EtoAc (5 mL × 3). The water layer was evaporated to dryness and then dissolved in MeoH. The solution was then subjected to silica gel TLC together with authentic d-glucose (R f , 0.12) and L-rhamnose (R f , 0.30). The plate was developed with CHCl 3 /MeoH/ H 2 o (13 : 7 : 2) lower phase, and visualised by anisaldehyde-H 2 So 4 solution (10 min at 105 °C).

DPPH radical scavenging activity
Each sample was dissolved in absolute MeoH and added to a DPPH solution (0.056 mg/mL, in absolute MeoH). After mixing gently for 30 min, optical densities were measured at 517 nm under 27 °C using a microplate reader (SpectraMax Plus 384, Molecular Devices, uSA). Vitamin C was used as the positive control.

Conclusions
In conclusion, eight flavonol glycosides (1-8) including a new prenylated flavonol glycoside (1) were isolated from the aerial parts of E. koreanum Nakai. Among the isolates, the non-prenylated kaempferol glycoside 8 showed good antioxidant activity. The results indicated that C-8 prenylation of the flavonol glycosides isolated from E. koreanum could reduce their antioxidant activities.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the National research Foundation of Korea Grant funded by the Korean Government [grant number NrF-2012r1A1A4A01009908].