Investigation of Flavonoid Glycosides from Neolitsea sericea var. aurata via the General Method and HPLC-SPE-NMR Sio-Hong Lam ( ), Chien-Kuang Chen ( ), Jeng-Shu Wang ( ) and Shoei-Sheng Lee* ( ) School of Pharmacy, College of Medicine, National Taiwan University, 1 Jen-Ai Rd., Sec. 1, Taipei 10051, Taiwan, R.O.C. Six flavonoid rhamnosides (1-2, 4-7) were separated from the water-soluble non-alkaloidal portion of the EtOH extract of the leaves of Neolitsea sericea var. aurata using various chromatographic techniques including Sephadex LH-20, centrifugal partition chromatography, and RP-18 Lobar. One additional com- pound (3) besides these six was identified by application of HPLC-SPE-NMR in a flavonoid rich fraction. The latter approach consumed only 1.5 mg of samples, theoretically equivalent to 0.2 g of dry leaves. This study demonstrates that HPLC-SPE-NMR has great advantage over the general methods on the aspects of manpower, research time, amounts of plant materials, and consumables in separation and characterization of natural products. Keywords: Neolitsea sericea var. aurata; Flavonoid glycosides; General methods; HPLC-SPE-NMR. INTRODUCTION Neolitsea sericea (Blume) Koidz. var. aurata Hayata (Lauraceae) is distributed on islands of the eastern Asia- Pacific Ocean ridge. 1 The Neolitsea plants were found to be rich in alkaloids and sesquiterpenes. 2,3 Flavonoids, how- ever, were rarely reported. We reported recently the minor N-oxides of aporphines, benzylisoquinolines and mor- phinans, along with the free bases from the ethanolic ex- tract of the leaves of the title plant. 4 During that study, we found that the water-soluble fraction of the same extract contained flavonoid glycosides. Hence, we continued to in- vestigate the constituents in this polar fraction. In this study, two approaches were used. One was using common chromatographic methods and the other was applying the high-performance liquid chromatography-solid phase ex- traction-nuclear magnetic resonance (HPLC-SPE-NMR) technique. The latter hyphenated technique as depicted in the figure was introduced in 2001 and has been demon- strated to be a powerful tool in phytochemical analysis. 5,6 In brief, each compound containing eluant from a reversed phase HPLC separation, monitored by photodiode array detector (DAD), was loaded on a respective SPE cartridge, whose capacity was enhanced by adding water to the eluant via a make-up pump. Then the loaded cartridges were flushed by nitrogen gas to dryness. The compound in the dry cartridge was then washed with deuterated solvent di- rectly into an inverted NMR flow probe, where all the 1 H detected NMR spectra were measured. This article de- scribes the outcome of our effort in applying these two approaches in disclosing the flavonoid glycosides from the leaves of the title plant. RESULTS AND DISCUSSION The fraction of flavonoid glycosides present in N. sericea var. aurata was obtained by passing the water solu- ble fraction (pH 4.0), obtained after removing alkaloids from the acidic aqueous extraction of the EtOH extract, through an Amberlite column, washed with methanol. The flavonoid glycosides in this fraction were then concen- trated via a Sephadex LH-20 column, separated via centrif- ugal partition chromatography and RP-18 Lobar columns. Such approaches led to the characterization of six com- pounds (1-2, 4-7). Their structures were elucidated mainly based on 1 H and 13 C NMR, and circular dichroic (CD) spec- tral analyses. They were identified as dihydroquercetin 3- O-rhamnopyranosides [1 (2S,3S), 2 (2R,3R), 4 (2S,3R) and 5 (2R,3S)], 7,8 quercetin 3-O-rhamnopyranoside (6), 9 and kaempferol 3-O-rhamnopyranoside (7). 10 Compounds 1, 2, Journal of the Chinese Chemical Society, 2008, 55, 449-455 449 * Corresponding author. Tel: +886-2-23123456 ext. 8392; Fax: +886-2-23916127; E-mail: shoeilee@ntu.edu.tw