Purification of a fucoidan from kelp polysaccharide and its inhibitory kinetics for tyrosinase

Highlights

• A new fucoidan from kelp crude polysaccharide was purified.

• GPC–HPLC profiles showed KPS-2-1 was a single component.

• The FT-IR spectrum showed that the purified fucoidan was a sulfated polysaccharide.

• The purified fucoidan is a strong competitive inhibitor of the tyrosinase.

• The study lays a good foundation for further developing the fucoidan from kelp.

Abstract

High-speed countercurrent chromatography (HSCCC) was used to separate kelp polysaccharide. HSCCC was performed using an aqueous two-phase solvent system composed of PEG1000–K₂HPO₄–KH₂PO₄–H₂O (0.5:1.25:1.25:7.0, w/w) by eluting a lower aqueous phase at 2.0 mL/min at 600 rpm, yielding two separate fractions, KPS-1 and KPS-2. The KPS-2 fraction was further purified by DEAE-Sepharose fast flow anion-exchange column chromatography to provide 3 fractions, KPS-2-1, KPS-2-2 and KPS-2-3. GPC–HPLC analysis indicated that KPS-2-1 fraction was a purified fucoidan. FT-IR analysis showed that KPS-2-1 was a sulphated polysaccharide. An analysis of enzymatic kinetics showed that the purified fucoidan was a competitive inhibitor of tyrosinase toward l-tyrosine, and the inhibitory constant K_i obtained from double-reciprocal plots was 0.9907 mg/mL.

Introduction

Fucoidan is a complex sulphated polysaccharide that is mainly derived from marine brown seaweed. Usually, fucoidan contains large proportions of l-fructose and sulphate (Bilan et al., 2006, Duarte et al., 2001). Previous studies have shown that fucoidan has a wide variety of bioactivities, such as inflammatory modulation and anti-virus and anti-tumor activities (Alekseyenko et al., 2007, Chandía and Matsuhiro, 2008, Cumashi et al., 2007, Hayashi et al., 2008, Yang et al., 2009). Recently, a fucoidan from algae Fucus vesiculosus has been demonstrated to inhibit human immunodeficiency virus (HIV) in vitro and shows a synergistic effect with azidothymidine (Chotigeat et al., 2004, Sugawara et al., 1989). Brown seaweed kelp (Laminaria japonica) is one of the most important economic seaweeds cultured in China, Japan and Korea, and is widely consumed as a marine vegetable in these countries (Gao et al., 2006, Pang et al., 2007, Suzuki et al., 2006). The use of kelp as a drug and an effective component of cosmetics have been well documented in traditional Chinese medicine (Zhang et al., 2007). Kelp is also used as manure, cattle feed, and food for human consumption, as well as a source of phycocolloids, such as agar, alginic acid and carrageenan (Chapman, 1970).

High-speed countercurrent chromatography (HSCCC) has been widely used for the separation and purification of natural products (Yang et al., 2009). HSCCC is a liquid chromatographic technique that operates under gentle conditions and allows non-destructive isolation, even for labile natural compounds. Due to the absence of any solid stationary phase, adsorptive losses are minimized, guaranteeing a 100% sample recovery (Scharnhop & Winterhalter, 2009). To date, the use of HSCCC to isolate products has focused on small organic compounds, and very few efforts have been made to apply this technique to polysaccharides and fucoidans.

Tyrosinase (EC 1.14.18.1), also known as polyphenol oxidase (PPO) (Burton, 1994, Fox, 1991, Robinson and Eskin, 1991, Sanchez-Amat and Solano, 1997, Wong, 1995), is a copper containing mixed-function oxidase widely distributed in microorganisms, animals and plants. This oxidase catalyzes two distinct reactions of melanin synthesis, the hydroxylation of a monophenol and the conversion of an o-diphenol to the corresponding o-quinone (López-Serrano et al., 2004, Lontie, 1984, Swan, 1974). The hydroxylation of l-tyrosine, the initial step in melanin synthesis, is of considerable importance since it is also the initial step in catecholamine synthesis. Alterations in melanin synthesis occur in many disease states. Melanoma specific anticarcinogenic activity is also known being linked with tyrosinase activity (Prezioso, Epperly, Wang, & Bloomer, 1992). Melanins are also found in the mammalian eye and brain. Tyrosinase may play a role in neuromelanin formation in the human brain and could be central to dopamine neurotoxicity as well as contribute to the neurodegeneration associated with Parkinson's disease (Xu et al., 1997). Tyrosinase is also responsible for browning in plants and considered to be deleterious to the color quality of plant derived foods and beverages. This unfavorable darkening from enzymatic oxidation generally results in a loss of nutritional and economic values and has been of great concern (Friedman, 1996, Jang et al., 2002, Lin et al., 2010). Similarly, the unfavorable browning caused by tyrosinase on the surface of seafood products has also been of great concern (Kajiwara et al., 2006, Kamkaen et al., 2007, Ogawa et al., 1984). Therefore, the regulation of the tyrosinase activity by its inhibitors has been the hot topic in recent ten years due to its potential applications in medicine, cosmetics and agriculture. To date, many different tyrosinase inhibitors have been suggested as being potential candidates for whitening agents or bio-insecticides (Huang et al., 2009, Kim and Uyama, 2005, Kubo et al., 1994, Nerya et al., 2003). Thus, it is interesting to investigate if kelp fucoidan or polysaccharide can become a potential tyrosinase inhibitor for the above-mentioned use.

In the present study, HSCCC in combination with DEAE-Sepharose fast flow (F.F.) anion-exchange chromatography was used to purify a fucoidan from kelp polysaccharide. GPC–HPLC and FT-IR spectrum analysis was performed to analyze the characterization of the purified fucoidan. The inhibitory kinetics of the fucoidan for tyrosinase toward l-tyrosine was also investigated.