Pharmaceutical Biology, 2010; 48(4): 422–426
RESEARCH ARTICLE
Hypolipidemic effect of fucoidan from Laminaria japonica in hyperlipidemic rats Ling Huang1, Kewei Wen2, Xuan Gao2, and Yonghong Liu2 Department of Oncology, Guangdong Provincial People’s Hospital, Guangzhou, China, and 2Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China
1
Abstract In this study, we investigated the effect of fucoidan polysaccharide sulfuric acid ester (FPS) from Laminaria japonica Aresch (Laminariaceae) on hyperlipidemic rats. FPS notably reduced the concentration of serum total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) of hyperlipidemic rats and increased the concentration of high-density lipoprotein cholesterol (HDL-C) and the activities of lipoprotein lipase (LPL), hepatic lipoprotein (HL), and lecithin cholesterol acyltransferase (LCAT). Keywords: Fucoidan; hyperlipidemic rats; hypolipidemic effect; Laminaria japonica Aresch
Introduction Atherosclerosis is a chronic inflammatory response in the walls of arteries. It is the major cause of heart disease, stroke, and death in the world. It is well established that elevated blood lipid levels constitute the primary risk factor for atherosclerosis (Harnafi et al., 2008). Many studies have shown that increased cholesterol concentrations in plasma cause coronary atherosclerosis. Recently, there has been more research on reducing and/or regulating serum cholesterol and triglyceride levels; in particular, dietary plants with cholesterollowering activity are considered useful in preventing disorders such as atherosclerosis (Ghule et al., 2006; Lemhadri et al., 2006; Kim et al., 2008). The brown seaweed, Laminaria japonica Aresch (Laminariaceae), a common seafood and important economic brown alga in China and many other countries, has been documented as a drug in traditional Chinese medicine for over 1000 years (Li et al., 2005).
It is considered effective in removing phlegm, inducing diuresis, alleviating edema, eliminating carbuncle, and clearing heat. L. japonica contains alginate, mannitol, laminaran, fucoidan, cellulose, protein, iodine (Podkorytova et al., 2007) and other mineral substances (Zhao et al., 2008), dibutyl phthalate (Namikoshi et al., 2006), etc. Sulfated polysaccharides in seaweed show diverse biological activities of potential medicinal value, such as anticoagulant (Nishino & Nagumo 1992), antitumor (Zhuang et al., 1995), contraceptive, antiviral (Feldman et al., 1999; Ponce et al., 2003), antioxidant (Qi et al., 2005; Tannin-Spitz et al., 2005; Wang et al., 2008), neuroprotective (Jhamandas et al., 2005), and hypolipidemic effects (Shanmugam & Mody, 2000; Veena et al., 2007). Fucoidan polysaccharide sulfuric acid ester (FPS), a group of sulfated heteropolysaccharides, extracted from L. japonica consists mainly of fucose and sulfate, with a smaller amount of arabinose and a trace of rhamnose. In this study, we investigated the effects of FPS, using a high-fat emulsion-fed hyperlipidemic rat model.
Address for Correspondence: Dr. Yonghong Liu, Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510-301, China. Tel: +86-20-89023244. Fax: +86-20-84451672. E-mail:
[email protected] (Received 13 October 2008; revised 19 January 2009; accepted 28 January 2009) ISSN 1388-0209 print/ISSN 1744-5116 online © 2010 Informa UK Ltd DOI: 10.3109/13880200903150435
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Fucoidan from Laminaria japonica 423
Materials and methods General experimental procedures Equipment included a type 722 ultraviolet (UV)-visible range microspectrophotometer (Unico (Shanghai) Instrument Co., Ltd.), a table-type high-speed refrigerated centrifuge (Beckman CS-15R; Beckman Instruments, Inc.), and an ultralow temperature freezer (Sanyo). Optical rotation was obtained using a Jasco DIP-370 digital polarimeter. Infrared spectra were measured on an Equinox 55 spectrometer (KBr). 1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AC 500 NMR spectrometer. High performance size exclusion chromatography (HPSEC) conditions were as follows: Waters Delta 600 system; Waters Millennium32 software; Waters refractive index instrument; Waters Ultrahydrogel 250 column (7.8 × 300 mm, 6 m); mobile phase: 0.1 M NaNO3, flow rate: 0.8 mL/min; eluates were monitored by the modified phenol–H2SO4 method. To estimate the molecular weight of FPS, Blue Dextran (type 2000) and several sizes of dextran sulfates were used as standards. High performance liquid chromatography (HPLC) conditions were as follows: Waters Delta 600 system; Waters Millennium32 software; Waters refractive index instrument; Hypersil NH2 column (4.6 × 250 mm, 5 m); mobile phase: MeCN:H2O/80:20, flow rate: 1.0 mL/min. Total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and highdensity lipoprotein cholesterol (HDL-C) assay kits were purchased from Biosino Bio-Technology and Science Incorporation (China). Total lipoprotein and hepatic lipases (LPL and HL) were obtained from Nanjing Jiancheng Bioengineering Research Institute (China). All chemicals and reagents, unless specified otherwise, were of analytical grade and were not purified, dried, or pretreated, and purchased from commercial sources. Plant material L. japonica was purchased at a local market in Qingdao in March 2003, and was authenticated by Professor Q. Chen at the South China Sea Institute of Oceanology, Chinese Academy of Sciences, China. A voucher specimen (No. 20030320) was deposited in an author’s laboratory (Y.L.). Preparation of FPS The finely powdered dry algae (particle size 50 mesh, 1 kg) were soaked for maceration in redistilled water (30 L) with added papain (1 g) in an extractor with stirring for 1 h at 60°C. The rate of agitation was ~60 rpm. The fermentation temperature was then increased to 90°C for 2 h, the mixture was centrifuged at 14,000 rpm,
and the supernatant was concentrated to 6 L by vacuum. Edible ethanol 95% (v/v) was added, and the final ethanolic concentration was 25% (v/v); 120 g MgCl2 was added, the mixture was left for 4 h, and then centrifuged at 14,000 rpm. Edible ethanol 95% (v/v) was added to the supernatant, and the final concentration of ethanol was 75% (v/v); this was left for 6 h, and then centrifuged at 14,000 rpm. The resultant precipitate was dried at 60°C, and distilled water was added to the residue (15:1, w/w). After the residue was dissolved, cation exchange resin (5:1, v/v) was added with stirring and the mixture left to stand for 3 h before filtering. The aqueous filtrate was then basified with NaOH to pH 6, the solution was dialyzed against distilled water for 2 h using 10,000 Da MW cutoff dialysis membranes, and then it was dried at 60°C (mean yield 1.3%) under reduced pressure. Hydrolysis of FPS The FPS (10 mg) was heated with 0.5 mL of 2 M trifluoroacetic acid for 2 h at 120°C. The acid was removed in vacuo by co-evaporations with distilled water, and the resulting syrup was treated with sodium borohydride in 3 mL of distilled water. The reduced material was analyzed by HPLC. l-Fucose, d-glucose, d-fructose, l-rhamnose, sucrose, and l-arabinose were used as standards. Sulfate was estimated turbidimetrically after hydrolysis of FPS in 2 M CF3COOH as above. Animals and treatments Male Sprague-Dawley (SD) rats, weight 180–220 g, were used, obtained from the Guangdong Provincial Medical Animal Center. All animals were maintained at a temperature of 25 ± 1°C, 55 ± 10% humidity, and 12 h on/off light cycle (7:00 a.m.–7:00 p.m.). The animals had unlimited access to water throughout the period of the study. They were allowed to acclimatize under climate-controlled conditions for a week before use. Animal experiments were conducted in accordance with current ethical regulations for animal care and use. With regard to changes in body weight, we found no statistically significant differences between the control and the sample-treated rats during the experimental period. Also, no cases of diarrheal symptoms were found. Sixty healthy male SD rats were randomized into six groups: normal group, control group, gypenosides (Gynostemma pentaphyllum (Thunb) Makino (Cucubitaceae), GP, 30 mg/kg)) group, and low-, moderate-, and high-dose FPS groups (0.1, 0.2, and 0.4 g/kg, respectively). Each group was administered by gavage with same-volume solutions (distilled water for normal and control groups, treatments for the other groups). Except for the normal group, groups of rats received high-fat emulsion (10 mL/kg) by gavage to establish hyperlipidemic models.
424 Ling Huang et al. Reference drug Gypenosides tablets (Ankang Zhengda Pharmaceutical Shareholding Co. Ltd., China) were used as the reference drug (positive control). They were dissolved in distilled water prior to administration.
of differences in the mean values; values are expressed as mean ± SEM (standard error of the mean). Significant differences are indicated by p values.
Results and discussion
Experimental hyperlipidemic diet
Preliminary characterization of FPS
The high-fat emulsion diet was prepared using 20% crude fat, 10% cholesterol, 2% sodium tauroglycocholate, and 1% propylthiouracil.
The infrared spectrum of FPS showed the characteristic band of S=O stretching vibration at 1259 cm−1 and a sulfate absorption band at 848.5 cm−1 (C-O-S, secondary axial sulfate), and a relatively small shoulder at 821 cm−1 (C-O-S, secondary equatorial sulfate) indicated that the majority of sulfate groups occupied position 4, and only a minor part of the sulfate was located at positions 2 and/or 3 of fucopyranose residues. A band was found at around 1647 cm−1 due to the carbonyl group (Bilan et al., 2002, 2004; Ghosh et al., 2004). The sulfate content (28.1%) was determined by the turbidimetric method. Acid hydrolysis of FPS and analysis by HPLC showed the presence of fucose (59.85%), together with a smaller amount of arabinose (7.89%) and a trace of rhamnose (0.14%) residues. The highly negative value of the optical rotation []25D = −135.1° (c, 0.5, H2O) of the polysaccharide was indicative of the presence of -l-fucopyranosyl residues (Chandía & Matsuhiro, 2008). The 1H NMR spectrum (not shown) presented two signals ( 5.36 and 5.32) in the -anomeric region. At higher field, two signals ( 1.21 and 1.18) assigned to methyl groups of fucose residues were present (Bilan et al., 2002, 2004). Proximate molecular size and quantity of FPS was determined by HPSEC analysis; on the basis of calibration with dextrans, the apparent molecular mass of the major peak of FPS would be 76 kDa.
High-fat emulsion-fed hyperlipidemic rat model In this model, the animals were fed with a high-fat emulsion diet for 28 days. To identify the induction of hyperlipidemia, blood was collected from the tail vein and then assayed for serum lipid index using an assay kit. The rats were then divided into six groups of 10 animals each by cholesterol level. Test samples were given orally once a day for a period of 28 days. Blood sampling At the end of the experimental period, and after 12 h fasting, the rats were anesthetized with ethyl ether and killed by decapitation. Blood was collected from the neck wound and left at room temperature for 30 min; the blood samples were then centrifuged at 3000 rpm and 4°C for 10 min. The serum was stored at −70°C for later biochemical analysis. Measurement of serum lipid levels Levels of TC, TG, LDL-C, HDL-C, LPL, HL, and lecithin cholesterol acyltransferase (LCAT) in serum were determined by enzymatic colorimetric methods using commercial kits. Statistical analysis Statistical evaluation of the results was done using statistical software SPSS 10.0 to determine the significance
Effect of FPS on serum blood lipids in hyperlipidemic rats Table 1 shows the effects of FPS of L. japonica on the serum lipid levels of the experimentally induced hyperlipidemic rats; serum TC, TG, and LDL-C levels were lowered by the FPS.
Table 1. Effect of fucoidan polysaccharide sulfuric acid ester (FPS) on serum blood lipids in hyperlipidemic rats. Group Dose (g/kg) TC TG HDL-C Normal 1.72 ± 0.40*** 0.89 ± 0.29*** 0.46 ± 0.09*** Control 10.27 ± 1.71 4.95 ± 1.62 0.31 ± 0.06 GP 0.03 5.65 ± 1.32*** 3.42 ± 1.55* 0.40 ± 0.09* Low-dose FPS 0.1 5.55 ± 1.28*** 3.39 ± 1.39* 0.40 ± 0.09* Moderate-dose FPS 0.2 5.20 ± 0.99*** 3.10 ± 1.06** 0.41 ± 0.08** High-dose FPS 0.4 5.63 ± 1.42*** 3.65 ± 1.52 0.37 ± 0.09 Values are mean ± SEM (n = 10). *p
