Structure of Ulvan Isolated from the Edible Green Seaweed, Ulva pertusa

Ulvan, rhamnan sulfate, was extracted from the edible green seaweed, Ana-aosa (Ulva pertusa), which is grown on the coast of the Okinawa Islands. The yield of ulvan was 8.5% (W/W), and the total carbohydrates, uronic acid and sulfuric acid and ash contents were 67.3%, 23.8%, 19.7% and 22.6%, respectively. L-Rhamnose, D-xylose and D-glucose residues were identified by liquid chromatography, and their molar ratio was 4.0:0.1:0.3. D-Glucuronic and L-idulonic acid residues were also identified in molar ratio of 1.0:0.2. The NMR (13C and 1H) and methylation analysis revealed terminal β-D-glucruonic acid, terminal α-L-idulonic acid, 1,3-linked α-L-rhamnose, 1,4-linked α-L-rhamnose, 1,2,4-linked α-L-rhamnose, 1,3,4-linked α-L-rhamnose, 1,2,3,4-linked α-L-rhamnose and 1,3,4-linked β-D-xylose. The sulfate groups were attached at the C-2 and C-3 positions of the 1,4-linked α-L-rhamnose as well as C-3 of the 1,4-linked β-D-xylose residues. The chemical structure of the ulvan from Ulva pertusa was determined.

In the previous studies, we identified fucoidans from commercially cultured Okinawamozuku (Cladosiphon okamuranus) [28] and Itomozuku (Nemacystus decipiens) [29]. Specifically, a novel fucoidan which was substituted with an acetyl group from the commercially cultured brown seaweed, Cladosiphon okamuranus [28] [32] was identified and patented [39]. The acetyl fucoidan exhibits some biological activities, such as antitumor [40] and immune-enhancing abilities [41]. An over-sulfated acetyl fucoidan, the sulfate content of which was 32.8%, showed a significant antitumor activity in vitro [40]. The results suggested that the over-sulfated acetyl fucoidan was applicable as an anticancer drug. Recently, the chemical structure of the acetyl fucoidan was determined [41]. The acetyl fucoidan consists of 1,3-linked α-L-fucopyranosyl residues and a branched β-D-glucuronopyranosyl residue at C-2 on the main-chain. The sulfate and acetyl groups branched at C-4 on the main chain. We also isolated a rhamnan sulfate from commercially cultured Hitoegusa (Monostroma nitidum: green seaweed) in Okinawa Prefecture, Japan and determined its chemical structure [38]. The rhamnan sulfate consists of 1,3-linked α-L-rhamnopyranosyl residues that branch at C-2 with trisaccharide side chains, D-GlcA(1→2)α-L-Rha-(1→2)-α-L-Rha-(1→. The acetyl fucoidan is now used as a supplement in health food, food and cosmetic industries in the world.
Ulva pertusa, an edible green seaweed is widespread in the natural environment of Japan, South Korea and China. The seaweed contains water-soluble rhamnan sulfate, which is referred to as ulvan [42]. The chemical structure of part of the ulvan from Ulva pertusa has been reported [43] [44]. This paper describes the fine chemical structure of the ulvan from Ulva pertusa.

Materials
Ulva fertusa was collected from the coast of Tomishiro City, Okinawa, Japan on April, 2007. The algae were washed with tap water and air dried at 40˚C for 48 h before being ground into powder.
The sample (20 g) was suspended in distilled water adjusted to a pH of 3.0 with 0.05 M hydrochloric acid and stirred at 60˚C for 3 h to extract the polysaccharide. The extract was neutralized with a 0.01 M NaOH solution, and then centrifuged at 23,000 g at 4˚C for 20 min. The supernatant was filtered through Celite 545 (Nakarai, Japan). The filtrate was precipitated by adding 2 volumes of ethanol, and the resulting solid was dried in vacuo.
The crude polysaccharide was dissolved in distilled water and the solution was passed through Celite 545. The filtrate was deionized by passing through a cation exchange column composed of Amberlite 120A H + (Organo, Japan) and neutralized. The solution was subsequently lyophilized.

Chemical Procedures
The total carbohydrate and uronic acid content were determined with the phenol-sulfuric acid [45] and carbazolsulfuric acid [46] methods using L-rhamnose and D-glucuronic acid as standards, respectively. The purified polysaccharide (70 mg) was dissolved in distilled water (20 mL) and sulfuric acid was added to reach a final concentration of 1.0 M. The mixture was subsequently heated to 100˚C for 3 h. The hydrolysate was neutralized with BaCO 3 .

High-Performance Anion Exchange Chromatography Coupled with a Pulse Amperometric Detector (HPAEC-PAD)
The monosaccharides in the hydrolysate of the polysaccharide were identified using a HPAEC (DX-500, Dionex Co., CA, USA), fitted with a Carbopack PA1 column and a pulsed amperometric detector. The column was eluted at flow rate of 1 mL/min at 35˚C with 10 mM NaOH.

Determination of the Sulfate and Uronic Acid Contents
Purified polysaccharide (10 mg) was dissolved in distilled water and hydrochloric acid was added to reach a final concentration of 1.0 M. The mixture was heated at 100˚C for 3 h. The hydrolysate was allied to HPAEC (DX-500, Dionex Co., CA, USA), on a column (A-SC4) equilibrated with 1.7 mM NAHCO 3 + 1.8 mM NaCO 3 .
The chromatography was carried out at 35˚C using a flow rate of 1.0 cm/min.

Desulfation of the Polysaccharide
The aqueous solution of the purified polysaccharide was passed through a cation exchange chromatography column (Dowex 50 W, Dow Chemical Co.) and was neutralized with pyridine. Then it was dialyzed overnight. The dried sample was dissolved in a 90% DMSO+10% methanol solution and heated at 80˚C for 4 h under a N 2 atmosphere [38] [41]. Then the sample was dialyzed, and freeze-dried.

Infrared Spectrum and Specific Rotation of the Polysaccharide
The infrared spectrum of the polysaccharide was recorded in KBr discs using a spectrophotometer (FTS-3000; Bio-Rad Laboratories Inc., CA, U.S.A.) in transmittance mode from 4000 to 400 cm −1 .
The specific rotation was measured at 589 nm using a polarimeter (P-1010; JASCO Inc., Tokyo, Japan) at room temperature. The polysaccharide solution (0.2%) was prepared in distilled water.

Methylation Analysis
The polysaccharide (5 mg) was methylated according to the procedure described by Ciucanu and Kerek [47]. The obtained permethylated polysaccharide was subjected to complete acid hydrolysis using 2 M TFA (2 mL) at 120˚C for 2 h. The hydrolysate was dissolved in 1 M NH 4 OH (0.2 mL). DMSO (1 mL) containing 20 mg of NaBH 4 was added and the mixture was incubated at 40˚C for 90 min. Subsequently acetic anhydride (0.2 mL) was added to the mixture. Anhydrous 1-methylimidazole (0.2 mL) and acetic anhydride (1 mL) were then added, and the reaction mixture was incubated at ambient temperature for 10 min. After extraction with chloroform and washing with water, partially methylated alditol acetates were obtained.
The partially methylated alditol acetates were analyzed using a gas chromatograph (GC-14A; Shimadzu Corp., Kyoto, Japan) equipped with a flame ionization detector using a capillary column (DB-1: 40 m × 0.25 mm, J&W Scientific Inc., CA, U.S.A.). The injector and detector temperatures were 210˚C and 270˚C, respectively. After injection, the oven temperature was maintained at 150˚C for 5 min, and then raised at 5˚C/min to 250˚C. This temperature was maintained for 5 min. The identities of the peaks were confirmed using GC-MS (GCMS-QP 1000EX; Shimadzu Corp., Kyoto, Japan) under the same conditions.

Preparation of Polysaccharide
Ulva pertusa reached 12 -20 cm long having two cell layers thick and round blade. The collected seaweed was washed with tap water and then dried in air-dried oven at 40˚C for 24 h. A polysaccharide was prepared and purified as described in Materials and methods.

Chemical Components of the Polysaccharide
The yield of purified polysaccharide was estimated to be 21.7% of the algal dry weight after the first ethanol precipitation. The polysaccharide yield was reduced to 8.5% after purification based on the dried weight of algae. The polysaccharide was 67.3% (W/W) carbohydrates. The purified polysaccharide also contained 23.8%, 19.7% and 22.6% of uronic acid, sulfate and ash, respectively. The results are summarized in Table 1.
An anion exchange high-performance liquid chromatogram of the hydrolysate of the polysaccharide (Figure  1) showed that peaks 1, 2 and 3 were L-rhamnose, D-glucose and D-xylose in the molar ratio of 4.0:0.1:0.3. The result indicates that the polysaccharide isolated from Ulva pertusa is an ulvan [43]. D-Dlucuronic acid and L-idulonic acid was identified by anion exchange high-performance liquid chromatography in the molar ratio of 1.0:0.2, as shown in Figure 2.

Specific Rotation and Infrared Spectrum (IR) of the Polysaccharide
The specific rotation [α] 589 of the ulvan (0.2% in water) at 25˚C showed a value of −0.006˚, indicating that α and β linkages were both involved.
In the IR spectrum of the ulvan presented in Figure 3, the major absorption at approximately 3400 cm −1 was attributed to the stretching of hydroxyl groups. Absorption at 2900 cm −1 resulted from C-H stretching of C-H groups. Absorption at 1638 cm −1 resulted from C=O of uronic acid. The absorption at 1250 cm −1 and two shoulder absorptions at 850 and 780 cm −1 were indicative of the presence of sulfate ester substitutions. The absorption at the former, 1250 cm −1 , was attributed to C-O-S stretching. The band at 1053 cm −1 corresponds to the stretching of the C=O of uronic acid and the vibration of the C-O-C bridge of the glucosides [43].
The 1 H spectra of the native and desulfated ulvan are presented in Figure 5(a) (37˚C) and  (Figure 5(b)). From a published paper [38], signal F (5.18 ppm) was assigned to be α-1,3-linked α-L-rhamnose. The results are presented in Table 3.

Methylation Analysis
The native and desulfated ulvan was methylated according to the procedure described by Ciucanu & Kerek [47]. The obtained permethylated ulvan was subjected to complete acid hydrolysis to furnish mixtures of the methylated sugars which were analyzed as the corresponding alditol acetates using gas-liquid chromatography (GC) and combined gas-liquid chromatography/mass spectroscopy (MS). The chromatograms are shown in Figure  6(a) (native ulvan) and Figure 6(b) (desulfated ulvan). Partially methylated alditol acetates were identified using published data [53] [54]. For the native ulvan (Figure 6(a) (9). After desulfation (Figure 6(b)), peaks 4 and 6 increased, while peaks 8 and 9 disappeared. The results indicated that sulfate groups were attached at C-2 and C-3 of L-rhamnose as well as C-3 of the D-xylose residue. The results are summarized in Table 4.

Discussion
Based on the NMR and methylation analysis of the ulvan as well as the chemical shifts reported in the literatures [48]- [52], residue A was assigned as terminal β-D-glucuronic acid, residue B as terminal α-L-idulonic acid, residue C as 1,4-linked α-L-rhamnose substituted side-chain, residue D as 1,4-linked α-L-rhamnose, residue E as 1,4-linked β-D-xylose and residue F as 1,3-linked α-L-rhamnose. After desulfation, peak 9 in Figure 5(a) disappeared in Figure 6(b), suggesting that sulfate groups were attached at C-2 and C-3 of the L-rhamnose residue on the main-chain. Furthermore, a sulfate group was attached at C-3 of the D-xylose residue because 2-mono-O-methyl-D-xylose (peak 8 in Figure 6(a)) disappeared, but 2,3-di-methyl D-xylose (peak 6) increased after desulfation (Figure 6(b)). The residues of peaks 5 (2-mono-L-rhamnose) and 7 (3-mono-L-rhamnose) remained even after desulfation, suggesting that terminal sugars (D-glucuronic acid and L-idulonic acid) are substituted at C-2 and/or C-3 of the L-rhamnose residues. Consequently, the chemical and structural results showed that the ulvan extracted from U. pertutsa consisted

Conclusion
Thus, on the basis of the results and discussion, we proposed the chemical structure of the ulvan (octa-saccharide repeating units) isolated from Ulva pertusa, as shown in Figure 7.