Je-Seung Jeon

Determination of major phlorotannins in Eisenia bicyclis using hydrophilic interaction chromatography: Seasonal variation and extraction characteristics

In this study, a hydrophilic interaction chromatography (HILIC) condition was developed for the simultaneous determination of five major phlorotannins from an extract of Eisenia bicyclis (Kjellman) Setchell with good linearity (r(2)>0.999). Based on this method, the seasonal variations and extraction characteristics, in terms of total extraction yield and the content of the phlorotannins, were investigated under various extraction conditions. In results, the yields and phlorotannins were increased two-to-four times in summer (June-October) and then, were decreased to normal levels in winter (November-March). In the extraction of E. bicyclis, ethanol percentage in water, extraction time and washing time significantly affected the yield of the extract and the phlorotannins, whereas the temperature and the sample/solvent ratio impacted the extraction to a lesser degree. These results will be useful information in the application of this macroalga in the commercial areas related to nutraceuticals, pharmaceuticals, and cosmeceuticals.

Content of antioxidative caffeoylquinic acid derivatives in field-grown Ligularia fischeri (Ledeb.) Turcz and responses to sunlight.

Ligularia fischeri (Ledeb.) Turcz, a commercial leafy vegetable, contains caffeoylquinic acid derivatives (CQAs) as major phenolic constituents. The HPLC chromatograms of leaf extracts collected from different areas in Korea showed a significant variation in CQA amount, and two tri-O-caffeoylquinic acids (triCQAs) were purified and structurally identified by NMR and MS from this plant. Radical scavenging activities among CQAs were found to be increased in proportion to the number of caffeoyl groups. Since this plant prefers damp and shady growth conditions, the effects of sunlight were investigated by growing plantlets in sunlight and shade for four weeks. Greater leaf thickness and higher phenolic contents were found for leaves grown in sunlight than in shade. Four major CQAs-5-mono-O-caffeoylquinic acid (5-monoCQA), and 3,4-, 3,5-, and 4,5-di-O-caffeoylquinic acid (diCQA)-were induced by solar irradiation, whereas the content of these compounds decreased steadily in shade leaves. The leaves of L. fischeri clearly showed adaptation responses to sunlight, and these characteristics can be exploited for cultivation of this plant for potential use as a nutraceutical and functional food.

Tyrosinase inhibition by water and ethanol extracts of a far eastern sea cucumber, Stichopus japonicus.

BACKGROUND Tyrosinase plays a key role in hyperpigmentaion and enzymatic browning. The present study was aimed at investigating the inhibitory effects of water and 70% aqueous ethanol extracts of Stichopus japonicus, a sea cucumber long consumed as a tonic food and traditional medicine, on the diphenolase activity of tyrosinase. RESULTS In the tyrosinase inhibition study, high-performance liquid chromatography completely separated L-3,4-dihydroxyphenylalanine and dopachrome from other compounds present in the extracts, and provided more reliable results than the commonly used spectrophotometry. The ethanol extract (IC(50)=0.49-0.61 mg mL(-1)) showed higher inhibitory activity than the water extract (IC(50)=1.80-1.99 mg mL(-1)). Enzyme inhibition by the extracts was reversible and of mixed type. For both extracts, the dissociation constants for binding to free enzyme were significantly smaller than those for binding to enzyme-substrate complex. Ethyl-α-D-glucopyranoside (IC(50)=0.19 mg mL(-1)), isolated for the first time from sea cucumber, and adenosine (IC(50)=0.13 mg mL(-1)), were identified as key tyrosinase inhibitors. CONCLUSION The sea cucumber extracts were demonstrated to possess considerable inhibitory potency against the diphenolase activity of tyrosinase, suggesting that the sea cucumber may be a good source of safe and effective tyrosinase inhibitors.

Preparative separation of sesamin and sesamolin from defatted sesame meal via centrifugal partition chromatography with consecutive sample injection

A preparative separation method using consecutive sample injection centrifugal partition chromatography (CPC) was developed to obtain sesamin and sesamolin from defatted sesame meal extracts. A two-phase solvent system consisting of n-hexane-ethyl acetate-methanol-water (8:2:8:2, v/v) was applied in reversed-phase mode (descending mode). Preliminary experiments with an SCPC-100 (column volume: 100mL) were performed to select the appropriate two-phase solvent system and sample injection times; these parameters were then used with an SCPC-1000 (column volume: 1000mL) in a 10-fold scale-up preparative run. A sample containing 3g of crude extract was consecutively injected four times onto the SCPC-1000, which yielded 328mg of sesamin and 168mg of sesamolin. These compounds were analyzed by high-performance liquid chromatography and determined to have purities of 95.6% and 93.9%, respectively. Sesamin and sesamolin (30μM) increased antioxidant response element (ARE) luciferase activity 2.6-fold and 1.9-fold, respectively.

Preparative Isolation of Antioxidant Flavonoids from Small Black Soybeans by Centrifugal Partition Chromatography and Sequential Solid-Phase Extraction

Unlike other members of the leguminous family that are generally used as food, Rhynchosia volubilis, a small soybean with a black seed coat, is used as a folk medicine in Korea. Using an on-line high performance liquid chromatography-2,2ʹ-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation screening system, one peak in the ethyl acetate fraction exhibited outstanding antioxidant capacity. To purify and determine the antioxidant compounds associated with this peak, we developed a centrifugal partition chromatography (CPC) method. The ethyl acetate fraction (500 mg) was subjected to CPC using a two-phase solvent system of n-hexane–ethyl acetate–ethanol–water (0.1:8:2:7, v/v) with a dual-mode CPC (descending-ascending order) in a bid to separate glycitin (3.1 mg), daidzin (14.4 mg), 6ʹʹ-O-acetyl genistin (6.9 mg), 6ʹʹ-O-acetyl daidzin (5.3 mg), and a mixture of epicatechin and genistin. The mixture was further separated through solid-phase extraction to yield epicatechin (6.8 mg) and genistin (23.5 mg), respectively. Structural identification of the R. volubilis flavonoids was performed by NMR and ESI-MS.

Rapid Detection of Antioxidant Flavonoids in Azalea (Rhododendron mucronulatum) Flowers using On-line HPLC-ABTS+ System and Preparative Isolation of Three Flavonoids by Centrifugal Partition Chromatography

High-performance liquid chromatography coupled to an on-line ABTS+-based assay (on-line HPLC-ABTS+) system was used to determine the principle antioxidants in azalea flowers. Three flavonoids, myricetin, quercetin, and kaempferol, recovered in ethyl acetate extracts of azalea flowers were determined to have antioxidant activities. These three flavonoids were isolated and purified by successive centrifugal partition chromatography (CPC) using two different biphasic solvent systems, consisting of n-hexane-ethyl acetate-methanol-water (3:5:3:5 or 4:5:4:5, v/v). A total of 46.2 mg of myricetin, 28.9 mg of quercetin, and 10.6 mg of kaempferol with purities of 97.0%, 95.4%, and 93.9%, respectively, were purified from 500 mg of ethyl acetate soluble material from azalea flowers. The structures were identified by their retention time, UV spectra, and ESI-MS in the positive ion mode and were confirmed by NMR experiments.

PREPARATIVE ISOLATION AND PURIFICATION OF PRENYLATED ISOFLAVONOIDS FROM CUDRANIA TRICUSPIDATA FRUITS USING CENTRIFUGAL PARTITION CHROMATOGRAPHY

Prenylated isoflavonoids such as 6,8-diprenylorobol, 6,8-diprenylgenistein and 4′-O-methylalpinumisoflavone were purified from n-hexane extract of C. tricuspidata fruits using centrifugal partition chromatography (CPC) with a n-hexane-ethyl acetate-methanol-water (7:3:6:4, v/v, ascending mode) solvent system in a one-step within 200 min. One-step preparative CPC yielded 22.4 mg of 6,8-diprenylorobol and 26.7 mg of 6,8-diprenylgenistein with 95% and 85% purity, respectively, from 350 mg of crude hexane extract. Further purification by crystallization in methanol yielded 4′-O-methylalpinumisoflavone (9.3 mg) at over 95% purity based on the HPLC analysis. The chemical structures of purified compounds were elucidated by 1H, 13C NMR, and ESI-MS.

Preparative Isolation of Polar Antioxidant Constituents from Abies koreana Using Centrifugal Partition Chromatography Guided by DPPH•-HPLC Experiment

Preparative separation of antioxidant constituents from the leaves of Abies koreana Wilson (Pinaceae) was performed by centrifugal partition chromatography (CPC) with a two-phase solvent system of ethyl acetate-isopropanol-water (9:1:10, v/v) target-guided by DPPH•-HPLC experiment. In DPPH•-HPLC experiment, trans-piceid exerted antioxidant activity, which was purified by Diaion-HP 20 macroporous resin chromatography and subsequent CPC. Maltol (98.3 mg), dihydrokaempferol 7-O-β-D-glucopyranoside (230.4 mg), and trans-piceid (165.9 mg) were purified from 2.19 g of crude extract with the purity over 95% by HPLC analysis. The chemical structures of isolated compounds were determined by 1H, 13C NMR, and ESI-MS.

Phytoestrogenic activity of Aceriphyllum rossii and rapid identification of phytoestrogens by LC–NMR/MS and bioassay-guided isolation

Aceriphyllum rossii Engler (Saxifragaceae) has been used as a nutritious food in Korea, but only minimal studies on this plant have been undertaken. We have examined the estrogenic effect of A. rossii and have discovered its potent constituents. To identify the estrogenic activity of A. rossii, we measured the transactivation of the estrogen-responsive element (ERE) in MCF-7 cells transfected with an ERE-containing construct after treatment of the extract and fractions of A. rossii. A. rossii showed a potent estrogenic activity and gallic acid ethyl ester; quercetin and kaempferol were identified as major constituents of the active fractions by liquid chromatography–nuclear magnetic resonance spectroscopy/mass spectrometry. We confirmed quercetin and kaempferol are the major active constituents after activity-guided isolation of seven compounds: gallic acid ethyl ester (1), quercetin (2), kaempferol (3), quercetin-3-O-(6″-galloyl)-β-d-glucopyranoside (4), quercetin-3-O-β-d-glucopyranoside (5), kaempferol-3-O-(6″-galloyl)-β-d-glucopyranoside (6), and kaempferol-3-O-β-d-glucopyranoside (7). These results show A. rossii could be a possible candidate for the treatment of postmenopausal symptoms.

PREPARATIVE ISOLATION AND PURIFICATION OF FLAVONOIDS FROM PTEROCARPUS SANTALINUS USING CENTRIFUGAL PARTITION CHROMATOGRAPHY

A centrifugal partition chromatography (CPC) was applied to separate and purify flavonoids from the extract of Pterocarpus santalinus. Three flavonoids, taxifolin (21 mg), dihydrokaempferol (18 mg), and naringenin (16 mg) were successfully separated from 500 mg of ethyl acetate fraction of P. santalinus using centrifugal partition chromatography with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (4:5:4:5, v/v) in single CPC run. Eryodictyol (8 mg) and quercetin (5 mg) were further purified by HPLC. The purities of the isolated compounds were determined to be over 90% by HPLC analysis and their structures were elucidated by 1 H NMR, 13 C NMR, and ESI-MS.