Isolation of Fucosterol from Pelvetia siliquosa by High-speed Countercurrent Chromatography

Abstract

We report here the use of high-speed countercurrent chromatography (HSCCC) in the preparative isolation and purification of the bioactive component, fucosterol, from Pelvetia siliquosa. A crude extract was obtained by ultrasonic extraction of powdered P. siliquosa using methylene chloride and was then subjected to separation and purification by HSCCC, coupled with evaporative lightscattering detection. Preparative HSCCC was performed successfully using a two-phase solvent system, n-heptane:methanol (3:2, v/v), to obtain 10.96 mg fucosterol with 96.8% purity from 50 mg of crude extract; the recovery rate was approximately 90.5%. Key words: Pelvetia siliquosa, Fucosterol, High-speed countercurrent chromatography, Evaporative light-scattering detection IntrodutionPelvetia siliquosa ("Tumbugi" in Korean) belongs to the family Fucaceae. It is a marine alga endemic to the Korean peninsula and grows on the craggy surfaces of the southern seashores (Yoon, 1995). It has been used traditionally similarly to seasoned sea greens for religious services and as a health food (Oh et al., 1990).Results of our previous studies indicate that the ether fraction of P. siliquosa exhibits hepatoprotective and glucoselowering effects, and fucosterol isolated from P. siliquosa has antioxidant and antidiabetic activities (Lee et al., 2002(Lee et al., , 2003(Lee et al., , 2004b. Additionally, it has been reported that fucosterol decreases angiotensin-converting enzyme levels, with a reduction in the glucocorticoid receptor in endothelial cells and inhibits the lymphatic absorption of cholesterol in rats (Hagiwara et al., 1986;Ikeda et al., 1988). Thus, fucosterol is a major component responsible for the various activities of the algae. Fucosterol is usually isolated by extraction with a non-polar solvent and silica-gel column chromatography and is analyzed as the trimethylsilyl derivatives by gas chromatography. We have previously reported a method for the quantitative determination of fucosterol in marine algae to accurately determine their composition and thus generate fingerprints. The structural identification of fucosterol was performed with infrared spectrum (IR), electron impact-mass spectrum (EI-MS), 1 H NMR and 13 C-NMR with tetramethylsilane (TMS) as internal standard. This structure was also confirmed by comparing with the previously reported spectral data. Its structure is shown in Fig. 1 (Lee et al., 2004a).High-speed countercurrent chromatography (HSCCC) is a liquid-liquid partition chromatographic technique that has recently gained increased interest. This technique relies on the use of centrifugal force for the retention of the stationary phase. HSCCC is more advantageous than other liquid-liquid techniques because of the shorter separation time, wider range of possible solvent systems, and quantitative material recovery Hostettmann, 1994, 2006;Ito, 2005 Original ArticleFish Aquat Sci 15(3), [191][192][193][194][195] 2012 Fish Aquat Sci 15(3), [191][192][193][194][195] 2012 http://dx.doi.org/10.5657/FAS.20...

Isolation of Steroid Compounds from Suruhan (Peperomia pellucida L. Kunth) and Their Antimalarial Activity

“…The olefenic proton for H22 and H23 in compound 2 were absent. The 13 C NMR spectrum showed 29 carbons including an oxymethine carbon signal at δ 71.5 and two olefenic carbon at δ 122.3 and δ 115.5. the double bonded unsaturation at δ 146.9 and δ 115.5 was characteristics of fucosterol [15,16] and two methylene carbon signals were present at δ 35.2 and 25.6 for C22 and C23 confirmed by DEPT 135. If we compared with DEPT 135 for compound 2 then we confirmed that this compound was having six methyl (CH3) groups, ten methylene (CH2) groups, nine methane (CH) groups and four quaternary carbons (Cq) groups.…”

“…The IR spectrum (KBr) of isolated showed characteristic absorption frequencies at 3373.6 cm -1 (O-H stretching); and 1247 cm -1 indicated C-O bond vibrations. The C-C vibrations was at 1641 cm -1 ; other absorption peaks includes 1457.3 cm -1 (CH 2 ); vibration at 1381.6 cm -1 (O-H), 1038 cm -1 (cycloalkane) and 881.6 cm -1 [15][16][17]. The H NMR spectra of compound showed the presence of two methyl singlets at δ 0.71 and 0.98 ppm (H-18, H-19); three methyl doublets that appeared at δ 1.10, 0.84 and 1.17 ppm; and a methyl triplet at δ 1.08 ppm.…”

“…Other frequencies include 1625.8 cm -1 as a result C=C stretching however this band was weak at 1450.6 cm -1 was a bending frequency for cyclic (CH2) and 1366.7 cm -1 for C-H bending. The vibration at 923.5 cm -1 due to C-O stretching [15][16][17][18]. Compound 2 was identified as fucosterol by comparison the 1 H NMR and 13 C NMR spectra with those of compound 1.…”

Sterols from the brown alga Cystoseira foeniculacea: Degradation of fucosterol into saringosterol epimers

“…The chromatographic profile of this sterol fraction showed two main peaks which were separated and obtained in sufficient amounts, by repetitive chromatographic runs, for further NMR study. The complete analysis of the NMR data of the two sub-fractions along with a comparison with literature data allowed their structure to be elucidated as fucosterol (1) (Hwang et al, 2012) and an epimeric mixture of saringosterols (2 and 3) (Ayyad et al, 2003;Catalan et al, 1983;Huh et al, 2012). The duplicate signals (C-17, C-20, C-22 to C-24, C-27 to C-29) in the 13 C NMR spectrum of 2/3 were in accordance with the occurrence of the two C-24 epimers.…”

“…The final isolation of sterols from algal lipophilic extracts was mainly conducted by RP-C 18 HPLC (Hwang et al, 2012;Terasaki et al, 2009). In this work, a chromatographic method based on the use of a stationary phase with a smaller hydrophobic selectivity (RP-C 8 instead of RP-C 18 ) was developed: it allowed a quick and easy separation of the main steroidal components from algal extracts.…”