Chemical Constituents from Sargassum micracanthum and Antioxidant Activity

MATERIALS AND METHODS

Plant material: Sargassum micracanthum were collected in May 2006 from Chuja Island of Jeju, Korea. Voucher specimen number JBR-513 was deposited at the herbarium of Jeju Biodiversity Research Institute (JBRI). The samples were washed three times with tap water to remove the salt, epiphytes and sand attached to the surface and then carefully rinsed with fresh water and maintained in a medical refrigerator at -20°C. Then, the frozen samples were lyophilized and homogenized with a grinder prior to extraction.

General: The NMR spectra were recorded on a Brucker AVANCE 3 spectrometer at 500 MHz (¹H) and 125 MHz (¹³C), with the residual solvent CDCl₃ used as an internal standard for ³H (7.23) and for ¹³C (77.0). The FAB Mass spectra data were obtained from the Korea Basic Science Institute (Daegu) on a Jeol JMS 700 mass spectrometer. ESI mass spectra analysis was carried out on a Thermo LCQ-Fleet LC-MS/nMS. Column Chromatography (CC) separations were on silica gel 60 (70-230 mesh, Merck) and celite 545 (Celite Korea). The TLC was performed on commercial plates (silica gel and F₂₅₄, Merck). Compounds were detected by UV (λ = 254 and 366 nm) irradiation and/or with a methanolic solution of anisaldehyde, sulfuric acid and acetic acid (90:5:1 mL), followed by heating.

Isolation of fucosterol, sargaquinoic acid and sargachromenol: The algal powder (370 g) was extracted three times with 80% ethanol and filtered. The filtrate was then evaporated at 40°C to obtain the ethanol extract, which was subsequently sequentially solvent partitioned into Hexane, CH₂Cl₂, EtOAc, butanol and water-soluble extracts. The CH₂Cl₂ extract was fractionated using celite column chromathography (Hexane/CH₂Cl₂ 0:1, 10:1, 5:1, 2:1, 0:1, CH₂Cl₂/EtOAc 10:1, 5:1, 2:1, 0:1, methanol). The 0:1 Hexane/CH₂Cl₂ celite column fraction was subjected to repeated silicagel column chromatography (CH₂Cl₂/methanol 40:1) to yield compound 1(43 mg). The 5:1 Hexane/CH₂Cl₂ celite column fraction was also subjected to silica gel column chromatography (Hexane/EtOAc/methanol 3:1:0.1) to afford compound 2 (13.5 mg), 3 (22.8 mg). The structure of these compounds was identified by comparing the NMR spectral data with those in the existing literature.

DPPH radical scavenging assay: DPPH radical scavenging activity was measured using the method described by Nanjo et al. (1996). An ethanolic solution of 60 μL of each sample (or ethanol itself as the control) was added to 60 μL of DPPH (60 μmol L^-1) in ethanol. After mixing vigorously for 10 sec, the solution was transferred into a 100 μL Teflon capillary tube and fitted into the cavity of the ESR spectrometer (JES-FA machine, JOEL, Tokyo, Japan). The spin adduct was measured on an ESR spectrometer exactly 2 min later. The measurement conditions used were: central field 3475 G, modulation frequency 100 kHz, modulation amplitude 2 G, microwave power 5 mW, gain 6.3x10⁵ and a temperature of 298 K.

Hydroxyl radical scavenging assay: Hydroxyl radicals were generated by the Fenton reaction and reacted rapidly with nitrone spin trap DMPO; the resultant DMPO-OH adducts was detectable with an ESR spectrometer (Rosen and Rauckman, 1984). The ESR spectrum was recorded 2.5 min after addition of a phosphate buffer solution (pH 7.4) with 0.3 M DMPO 0.2 mL, 10 mM FeSO₄ 0.2 mL and 10 mM H₂O₂ 0.2 mL using an ESR spectrometer set at the following conditions: central field 3475 G, modulation frequency 100 kHz, modulation amplitude 2 G, microwave power 1 mW, gain 6.3x10⁵ and a temperature of 298 K.

Statistical analysis: The data are expressed as the Mean±SE. A statistical comparison was performed via a one-way Analysis of Variance (ANOVA) followed by Duncan’s multiple range test (Duncan, 1955). The p-values of less than 0.05 were considered to be significant.

RESULTS AND DISCUSSION

Brown algae of the genus Sargassum, (Sargassaceae, Fucales) are known to contain structurally unique secondary metabolites, such as plastoquinones, chromanols, a cyclopentenone and polysaccharides. These compounds show various biological activities due to their unique structure. We also previously reported that S. micracanthum showed strong anti-inflammatory activity. Since, the bioactive compounds, not only the structure-activity-relationship toward the development of new drugs or supplements, but also the biological functions in the algae themselves was still unclear, this study screened its antioxidant activity via its scavenging effect on DPPH free and hydroxyl radicals. As the CH₂Cl₂ fraction showed the strong DPPH radical scavenging activity of 83.1% at a concentration of 125 μg mL^-1. (IC₅₀ values, 45.3 μg mL^-1), the active compounds of this fraction was isolated and identified as sargaquinoic acid, sargachromenol and fucosterol, based on a comparison of NMR spectroscopic data and previous literature as follows Lee et al. (2003), Seo et al. (2004) and Cañabate-Díaz et al. (2007).

Table 1:	NMR spectroscopic data for sargaquinoic acid and sargachromenol

Compound 1 fucosterol: C₂₉H₄₈O, white amorphous powder, FABMS : positive mode m/z 395.5 (M+H-H₂O)⁺: 1H NMR (500 mHz, CDCl₃) δ_H (ppm): 5.33 (1H, br d, J = 5.0 Hz, H-6), 5.17 (1H, q, J = 6.5 Hz, H-28), 3.53 (1H, m, H-3), 1.55 (3H, d, J = 7.0 Hz, H-29), 0.98 (3H, s, H-19), 0.97 (3H, br s, H-21), 0.96 (3H, d, J = 1.2 Hz, H-27), 0.95 (3H, d, J = 1.5 Hz, H-26), 0.66 (3H, s, H-18); ¹³C-NMR (125 MHz, CDCl₃) δ_C (ppm): 146.9 (C-24), 140.7 (C-5), 121.7 (C-6), 115.5 (C-28), 71.8 (C-3), 56.7 (C-14), 55.7 (C-17), 50.1 (C-9), 42.3 (C-13),42.2 (C-4), 39.7 (C-12), 37.2 (C-1), 36.5 (C-10), 36.4 (C-20), 35.2 (C-22), 34.7 (C-25), 31.9 (C-7,8), 31.6 (C-2), 28.2 (C-16), 25.6 (C-23), 24.3 (C-15), 22.2 (C-26), 22.1(C-27), 21.0 (C-11), 19.4 (C-19), 18.7 (C-21), 13.1 (C-29),11.8 (C-18).

Compound 2 sargaquinoic acid: Brown viscous oil, ESI-MS/MS: negative mode m/z 424.06 (M-H)¯, 379.3 (M-COOH)¯: ¹H, ¹³C NMR Table 1.

Compound 3 sargachromenol: Brown viscous oil, ESI-MS/MS: negative mode m/z 424.03 (M-H)¯, 379.2 (M-COOH)¯: ¹H, ¹³C NMR Table 1.

Three compounds were then used in further experiments with an ESR spectrometer. The ESR spin trapping provides a sensitive, direct and accurate means of monitoring reactive species (Guo et al., 1999). Therefore, this study used ESR to compare the DPPH and hydroxyl radical scavenging abilities of the sargaquinoic acid, sargachromenol and fucosterol (Fig. 1A) isolated from S. micracanthum with those of a commercial antioxidant, such as ascorbic acid.

DPPH is a stable free radical donor that is widely used to test the free radical scavenging effect of natural antioxidants. The scavenging activity of the sargachromenol (SC) and sargaquinoic acid (SQ) towards DPPH free radicals is shown in Fig. 1B, where, 78.85 and 69.82% of the DPPH free radicals were scavenged at 250 μM, plus the radical scavenging occurred in a dose-dependent manner. Generally, hydroxyl radicals are generated from hydrogen peroxide and the scavenge hydroxyl radicals passed through the so-called Fenton reaction (Huang et al., 2002; Heo et al., 2008). Therefore, to scavenge hydroxyl radicals, the following in vitro reaction was generated: Fe²⁺+H₂O₂→Fe³⁺+¯OH+^●OH. As shown in Fig. 1B, the sargaquinoic acid, sargachromenol and fucosterol was not exhibited a decrease in the amount of DMPO-OH adducts by the ESR signals. In conclusion, as shown in Fig. 1C, sargachromenol is relatively strong DPPH antioxidant activity in three constituents. However, the sargachromenol exhibited relatively lower levels of DPPH free radical activity (IC₅₀ values, 49.3 μM) than the commercial antioxidant ascorbic acid (IC₅₀ values, 19.9 μM). Natural antioxidants can protect the human body from serious cellular or molecular damage by free radicals and retard the progress of many chronic diseases in food and cosmetic industry. Thus, owing to growing health concerns, interest has significantly increased in acid.

Fig. 1:	Chemical structures and antioxidant activities of fucosterol, sargachromenol and sargaquinoic

(A) Chemical structures of fucosterol (FU), sargachromenol (SC) and sargaquinoic acid (SQ). (B) DPPH and hydroxyl radicals scavenging activity of FU, SC and SQ. ESR spectrum obtained in an ethanol solution of 60 μmol L^-1 DPPH of three compounds and ascorbic acid. ESR spectrum obtained in Fenton reaction system of three compound and ascorbic acid. (C) DPPH Radical scavenging activities of SC and SQ via the ESR system. SC: Sargachromenol, SQ: Sargaquinoic acid, FU: Fucosterol, AS: Ascorbic acid finding natural antioxidants for use in food, cosmetic and medicinal materials to replace synthetic antioxidants with harmful effects. Even if, sargachromenol and sargaquinoic acid possessed relatively lower antioxidant activities than synthetic chemicals, therefore they may be a useful natural radical scavenger and a possible supplement for the food, pharmaceutical and cosmetic industries.

ACKNOWLEDGMENTS

This research was supported in part by the Program for the RIS (Regional Innovation System; JEJU SEA-GREEN PROJECT) and the Regional Technology Innovation Program (RTI04-02-07), both of which are managed by the Ministry of Knowledge and Economy, Korea.