Chemical structure of sulfated polysaccharides from brown seaweed (Turbinaria turbinata)

ABSTRACT

The chemical structure of three sulfated polysaccharides fractions (TtF1, TtF2, and TtF3) obtained from anion-exchange separation of aqueous extracts of brown seaweed (Turbinaria turbinate) were studied. The infrared spectra patterns showed that the fractions possess functional groups similar to that of sulfated polysaccharides. The sulfated polysaccharides fractions exhibited molecular weights of 223.5, 495.5, and 326.05 kDa, respectively, for TtF1, TtF2, and TtF3. ¹H NMR spectra of TtF2 and TtF3 contain α-anomeric protons (5–5.6 ppm), ring protons (3.4–4.4), and methyl protons (1–1.3 ppm) while that of TtF1 only exhibited ring protons and methyl protons. Rheological data were fitted to power law which revealed that the fractions were Newtonian and/or presented weak pseudoplastic behavior. Consistency values increased with concentration in all fractions. Consistency values of TtF2 were the highest, followed by TtF1 and then TtF3. Thermal degradation patterns of TtF1 and TtF2 were similar but different from that of TtF3. This study confirmed that chemical and physical characteristics of sulfated polysaccharides fractions are interrelated and provided in-depth understanding of sulfated polysaccharides of brown algae.

KEYWORDS:

• Sulfated polysaccharide
• Brown seaweed
• Rheology
• Thermogravimetric
• Fucoidan
• Alginate, Laminaran

Introduction

The cell wall of brown seaweeds contain sulfated polysaccharides (SPs), such as fucoidan, alginate, and laminaran, with varying biological properties.^[1] Numerous reports have shown that SPs exhibited structural variations due to factors like seaweed species, extraction methods, processing conditions, and environmental conditions.^[2–5] Structural variations can be in the form of position and degree of sulfation and/or monosaccharide composition and percentage of uronic acid.

In addition to the chemical characterization of SPs, efforts have also been made to elucidate physical properties of SPs. Both rheological and thermogravimetric analyses of SPs have been conducted. SPs solutions exhibited psuedoplastic or Newtonian fluid characteristics. Alginate extracted from Laminavia hyperborea and Macrocystis pyrfera at 1% concentration exhibited apparent viscosities of 0.03 and 0.35 Pa at 35°C, respectively.^[6] The apparent viscosity of 4.4% of alginate obtained from Lessonia nigrescens was 0.05 Pa at 30°C.^[7] Rioux and co-workers reported that out of four brown seaweed species investigated, Fucus vesiculosus exhibited the highest viscosity among fucoidan fractions and Saccharina longicruris exhibited the highest apparent viscosity among alginate fractions.^[8]

We hypothesize that SPs obtained from different species of brown seaweed will have different physical properties which might be due to their chemical properties. Therefore, relationships between chemical structure and physical properties of SPs obtained from brown seaweeds need thorough investigation. The focus of this work is to investigate the structural characteristics of sulfated polysaccharide extracted from T. turbinata of Malaysian origin. In order to elucidate the possibility of a relationship between chemical and physical properties, three SP fractions obtained from anion exchange chromatography were studied.

Methodology

Extraction and purification of SPs

The dried powdered of T. turbinata was hydrolyzed for 19.5 h with 0.15% cellulase at pH of 5. Enzyme activity was stopped by heating for 10 min at 100°C and the filtrate was collected. The solution was concentrated and subjected to ethanol precipitation (4:1 v/v; overnight at 5°C) to obtain crude polysaccharide extract. The crude polysaccharide precipitate was further purified using anion exchange chromatography (DEAE-cellulose, 17 × 2.5 cm column, eluted with 50 mM sodium acetate buffer (pH 5.0) solution containing 0, 0.2, 0.4, 0.8, and 1.2 M of NaCl). Fractions of 5 mL each were collected using a fraction collector and 100 μL of solution from each fraction was analyzed for total sugar using phenol sulfuric method.^[9] Solutions of fractions TtF1, TtF2, and TtF3, respectively, eluted with buffer containing 0.4, 0.8, and 1.2 M NaCl were obtained. Each solution was thoroughly dialyzed against fresh water for 3 days at 4°C under continuous stirring. All fractions where lyophilized and stored until needed.

Monosaccharide analysis

Samples were subjected to alditol acetate derivitization and the derivitized sugar were quantified by an Agilent (Santa Clara, CA) 7890 gas chromatography with flame ionization detector. SP^TM-2380 column (30 m × 0.25 mm × 0.2 μm, Supelco, Bellefonte, PA) was used for separation. The flow rate was kept constant at 0.8 mL/min and helium gas was the carrier. The injector, oven, and detector temperature were set to 230, 100, and 250°C, respectively.

Functional group analysis

Fourier transform infrared spectrometer (FTIR; Nicolet 8700 Thermo Scientific) equipment was used to obtain the IR spectrum of purified SPs. Approximately 1.5 mg of pure polysaccharide was ground with KBr and pressed to form pellet disc. The disc was placed in the sample compartment before the spectra was obtained. The frequency range used was between 4000–400 cm⁻¹. Sample spectra were subtracted from background spectra. Then the peaks were identified and labeled using OMNIC software by Thermo Scientific.

Molecular weight analysis

The molecular weights of the purified extracts were determined according to the method of Rioux, Turgeon, and Beaulieu^[8] with some modifications. Samples were dissolved in 0.1 μm filtered high-performance liquid chromatography (HPLC) grade water and filtered through 0.45 μm nylon syringe filters before analysis. The dn/dc value of 0.129 for SPs was used according to the determination of Rioux, Turgeon, and Beaulieu.^[8] The weight average molecular mass (M_w) of the polysaccharides were determined by high-performance size exclusion chromatography–multi-angle light scattering (HPSEC–MALS). The HPSEC system consisted of an Agilent 1200 HPLC with autosampler, heated column compartment, and differential refractive index (DRI) detector. The MALS detector was an 18 angle Wyatt Dawn Helios-II detector (Wyatt Technologies, Santa Barbara, CA) with a laser. Shodex SB-804 and SB-806M columns were used in sequence for separation. HPLC grade water which was filtered through 0.1 μm nylon membrane filter was used for the mobile phase and the flow rate was 0.50 mL/min. The column and DRI were set to 30°C and the MALS was run at room temperature. Data was processed using ASTRA software 6.0.5.3. The molecular weight was calculated using a second order Zimm plot.^[8]

¹H-NMR analysis

Sample preparation was carried out by dissolving 6 mg of freeze-dried pure polysaccharide in 400 μL of Deuterium water (D₂O). The solution was stirred for 2 h at 40°C and then freeze-dried. The process was repeated two times. The lyophilized samples were dissolved in D₂O (0.6 mL) a final time and placed in NMR tubes (8 in, 5 mm, thin wall). ¹H spectra were taken using a Bruker 400 MHz NMR. The analysis was conducted at 25°C for 64 scans.

Rheological analysis

Stock concentrations (2.0 %) of pure SPs fractions were prepared by dissolving 40 mg of samples in 2 mL of distilled water. Other three concentrations (0.25, 0.5, and 1.0%) were prepared by serial dilution of the 2.0% solution. The polysaccharide solutions were analyzed using a Stresstech controlled stress/strain rheometer (ATS Rheosystems, Bordentown, NJ) with a 2° cone and plate. The solutions were pipetted (0.3 mL) between the plates and evenly spread out. Flow tests were achieved at various share rates of 3 to 1000 s⁻¹ at 10 points per decade and running time of 5 s. Temperature of measurement was 23°C. Power law model (Eq. [4]) was used for curve modelization.

(1)

(2)

Substituting Eq. (2) into Eq. (1):

(3)

(4)

where γ represents share rate, η represents apparent viscosity, n represents flow behavior index, τ represents shear stress, and represents consistency index.

Thermogravimetric analysis

About 2–3 mg of freeze-dried SPs fractions were weighed into the sample compartment of the TGA Q500 V6.7 Build 203. The samples were heated from 40–800°C at a ramp rate of 25°C/min at a flow rate of 50 mL/min. The instrument measures percentage change in the weight of sample over the heating period. The equipment was allowed cool to for 30 min before the next sample was analyzed. Data analysis was done using a TA Universal analyzer.

Data analysis

The results were analyzed for significant differences using SPSS software for analysis of variance (ANOVA) and means were separated using Duncan’s multiple range techniques.

Results and discussion

Monosaccharide Composition of Purified Polysaccharides

The results of monosaccharide composition of the purified polysaccharides are presented in Table 1. Monosaccharide composition of the fractions varies considerably. This is in line with previous reports that about 35,600 species of algae already identified were of diverse cell wall components.^[10] All the fractions possess a very small amount of rahmnose, arabinose, and xylose. Fractions TtF1 and TtF2 contain high levels of mannose compared to TtF3. Fucose was highest in TtF3 followed by TtF1 and least in TtF2. Also, TtF3 predominantly contained the highest amount of galactose followed by TtF2 and then TtF1. The monosaccharide composition suggests that TtF1 and TtF2 are majorly laminaran because of high glucose and mannose while TtF3 is majorly fucoidan due to high fucose. Previous reports have shown that fucoidan from brown seaweed contain significant amount of fucose. Water soluble polysaccharide obtained from same genus of brown seaweed (T. conoides) reportedly contained 59% fructose,^[11] a very close value to 60.92% obtained in this work.

Table 1. Monosaccharide composition of sulphated polysaccharides obtained from T. turbinate.

Samples	Monosaccharide (%)
	Rahmnose	Fucose	Arabinose	Xylose	Mannose	Galactose	Glucose
TtF1	0.12±0.00^c	19.28± 0.00^b	0.50± 0.00^b	0.73± 0.00^b	41.86± 0.03^a	0.44± 0.00^c	35.53± 0.03^a
TtF2	0.19±0.00^b	8.32± 0.00^c	0.50± 0.00^b	0.41± 0.00^c	33.49± 0.09^b	9.75± 0.03^b	24.19± 0.03^b
TtF3	0.41±0.03^a	60.92± 0.09^a	0.62± 0.00^a	1.51± 0.00^a	4.46± 0.03^c	18.84± 0.03^a	13.18± 0.06^c

Values = mean ± standard deviation, n = 3.

Values in the same column with different letters are significantly different at p < 0.05.

FTIR analysis of purified polysaccharides

Figure 1 shows the IR spectra for the 3 polysaccharide fractions (TtF1, TtF2 and TtF3) and Table 2 showed the wave number with corresponding functional group. The spectra patterns show that the fractions possessed functional groups similar to that of SP. Equal intensity was observed for TtF3 and TtF2 spectra at 825–840 cm⁻¹ for C-O-S, while that of TtF1 was very low. Spectra of TtF3 gave higher peaks at 1220–1260 cm⁻¹ for S=O compared to spectra of TtF2 while spectra of TtF1 was the smallest. Hence, suggesting that sulfate ion composition of TtF3 might be the highest followed by TtF2 and TtF1. Histochemical studies of algal polysaccharides has shown presence of sulfate group according to FTIR reading. During the biosynthesis of polysaccharide in algae’s golgi body, modification with sulfate, pyruvate, and methyl groups occurred through sequential substitution reactions.^[12] Sulfate ions were rapidly incorporated into cellular polymer, including fucoidans of Fucus distichus.^[13] Sulfate ion was incorporated after 10 h of zygote development in Fucus sp.^[14,15]

Table 2. FTIR spectra of purified sulfated polysaccharide of T. turbinata in the frequency range 4000–400 cm.⁻¹

Samples	Frequency (cm^−1) of spectra
	1220–1260	825–840	3028–3423	2924.08	2350–2364.72	2121–2143	1635–1641
TtF1	1261.58	825.23	3450.52	2920.32	2355.34	2128.76	1635.28
TtF2	1259.72	820.31	3446.07	2920.32	2356.73	2125.32	1635.32
TtF3	1257.90	836.74	3455.32	2939.31	2363.06	2128.76	1635.79
Functional group	S=O	C-O-S	O-H	C-H	O-C-O or C-OH	C-H (fucose)	OH (Uronic)

Values = mean ± standard deviation, n = 3.

Values in the same column with different letters are significantly different at p < 0.05.

TtF1: sulfated polysaccharide fraction eluted with buffers that contained 0.4 NaCl in anion exchange chromatography.

TtF2: sulfated polysaccharide fraction eluted with buffers that contained 0.8M NaCl in anion exchange chromatography.

TtF3: sulfated polysaccharide fraction eluted with buffers that contained 1.2 M NaCl, in anion exchange chromatography.

Figure 1. FTIR spectra of sulfated polysaccharides obtained from T. turbinata. TtF1, TtF2, and TtF3 are sulfated polysaccharide fraction eluted with buffers that contained 0.4, 0.8, and 1.2 M NaCl, respectively, in anion exchange chromatography.

Molecular weight of purified polysaccharides

The molecular weights (M_w) of SP fractions were measured using a HPSEC-MAL-RI system to acquire RI and MAL signals shown in Fig. 2a–2c. According to Table 3, the M_w of TtF1, TtF2, and TtF3 were 223.5, 495.5, and 326.05 kDa, respectively. We compared our result with fucoidan, which is the most studied water soluble SP from brown seaweed. Among all the SP fractions, the Mw of TtF3 falls within the ranges previously reported for fucoidan fractions obtained E. cava (21–16,000 and 18–359 kDa).^[16,17] Other closer values of fucoidans M_w have also been reported. M_w of fucoidan from Ascophyllum nodosum, Saccharina longicruris, and Fucus vesiculosus are 417, 454, and 529 kDa, respectively.^[8] The variation in fucoidans M_w has been associated to processing conditions used during extraction of the biomolecules, different in species and seasonal variation.^[17]

Table 3. Molecular weight and polydispersity of sulfated polysaccharide fractions obtained from anion exchange chromatography of water soluble extract of T. turbinate.

Samples	Molecular mass moments (g/mol or Da)			Polydispersity
	Mw × 10^3	Mz × 10^3	Mn × 10^3	Mw/Mn	Mz/Mn
TtF1	223.50 ± 2.50^c	223.6 ± 0.03^c	223.1 ± 0.52^b	1.01	1.01
TtF2	495.50 ± 7.03^b	467.45 ± 8.20^b	413.76 ± 5.6^a	1.06	1.13
TtF3	326.05 ± 10.60^a	254.73 ± 2.8^a	166.49 ± 9.5^a	1.28	1.53

Values = mean ± standard deviation, n = 3.

Values in the same column with different letters are significantly different at p < 0.05.

TtF1: sulfated polysaccharide fraction eluted with buffers that contained 0.4 NaCl in anion exchange chromatography.

TtF2: sulfated polysaccharide fraction eluted with buffers that contained 0.8M NaCl in anion exchange chromatography.

TtF3: sulfated polysaccharide fraction eluted with buffers that contained 1.2 M NaCl, in anion exchange chromatography.

Figure 2. HPSEC-MAL-RI Chromatogram of different sulfated polysaccharide fractions from T. turbinata. A: TtF1; B: TtF2; and C: TtF3, LS: light scattering, RI: refractive index.

Extraction conditions have been reported to affect the M_w of polysaccharide obtained from plant and algae materials. Mild enzymatic extraction conditions resulted in large M_w of polysaccharide macromolecule (>10,000 kDa) while use of acid resulted to smaller molecules (710–5200 kDa). Proper dissolution of fucoidans might be achieved during extraction through heating due to breaking of hydrogen bond responsible for holding the polymer together.^[18] Marked reduction in M_w of fucoidans occurred, when other processing like boiling, microwave heating, ultrasonic, use of CuAc/H₂O₂, high pressure, agitation, etc., were employed (Synytsya et al., 2010).^[19] Yang et al.^[18] reported that boiling for 15 min resulted in reduction of M_w of fucoidans from 23,600 to 5,500 kDa; while heating in a microwave for 2 min caused reduction up to 500 kDa. This explains the reason for low molecular weight observed in our result. During extraction, our sample was subjected to a long boiling time (10 min).

Biosynthesis of polysaccharide does not follow any designated molecule template. Therefore, polysaccarhides of the same origin exist in a range of molecular weights and are said to be polydisperse. Polydispersity describes the existence of substances in a range of molecular weights rather than having a singular molecular weight. Most polysaccharides are polymolecular while all polysaccharides are polydisperse^[20] but at different extents. It is difficult to compare the numerical value of polydispersity with literatures because of lack of information.

¹H NMR of purified polysaccharides

Figures 3a–3c depict the ¹H NMR spectra of the three SPs fractions. The spectra of TtF2 and TtF3 show α-anomeric protons (5–5.6 ppm), ring protons (3.4–4.4), O-acetyl groups (~2.2 ppm) and methyl protons (1–1.3 ppm), while that of TtF1 only shows ring protons and methyl protons. Similar regions were reportedly observed in ¹H NMR spectra of fucoidan.^[2,21–23] The methyl proton has been previously assigned to C6 methyl proton of L-fucopyranose while the signal at α-anomeric proton is common with α-L-fucopyranosyl.^[23]

Figure 3. ¹H NMR spectra of sulfated polysaccharides extracted from T. turbinate.

The O-acetyl group observed in spectra TtF2 and TtF3 suggest that they contained acetylated polysaccharide. Previous reports have also shown presence of O-acetyl groups in fucoidan.^[21,22,24] Although acetyl groups were considered as an impurity, it was later confirmed to be part of the polysaccharide structure.^[24] Biosynthestic mechanism for O-acetylation of fucoidan has not been reported.^[12] However, study of model plant Arabidopsis revealed that reduced wall acetylation (RWA) and the trichome birefringence-like (TBL) proteins were involved in O-acetylation of plant polysaccharide.^[25]

The chemical shifts and bond descriptions were obtained using the Complex Carbohydrate Research Center (CCRC) online database. It revealed that the SPs structure comprised of β-L-arabinofuranosyl, α-L-arabinofuranosyl, α-D-Xylopyronose, β-DGlucopyronose, D-Glucitol, β-DGalactopyronose, and α-L-Fucopyronose. Previously, researchers have made efforts to relate the chemical shift in ¹H-NMR spectra with the location of glycosides bond. Signals at 5.10 and 5.18 ppm within the anomeric region have been associated with presence of α-3 linked and α-3, 4 linked L-fucopyranose residue as observed for fucoidan of Hazikia fusiform.^[22] Bilan et al.^[21] assigned the signal at the methyl proton to the presence of α-3-linked 2-mono-O-sulfated and α-3-linked unsulfated L-fucopyranose residues, while the signal at the ring proton was assigned to 4-linked 2-mono-O-sulfated L-fucopyranose residues. Farias et al.^[26] linked the signal at 4.61 ppm with the presence of 3-linked D-galactopyronosyl residue.

Presence of numerous overlapping signals in the ¹H-NMR was an indication that SPs were very complex polysaccharides in agreement with previous reports. This is in-line with previous findings that tried to elucidate the structure of fucoidan extracts from different seaweed.^[23,27] The most common facts regarding the structure of fucoidan from brown seaweed is that it consists of alternating (1 → 3) or (1 → 4) linked α-L-Fucp and β-D-galactopyranose (β-D-Galp), which is partially acetylated or sulfated at C2 and C4 position.^[28]

Rheological properties of purified SPs

In order to investigate the flow behavior of SPs fractions obtained from T. turbinata, flow tests were conducted and fitted with the power law. The flow curves of different concentrations of SPs fractions are shown in Fig. 4. The graphs show that the shear stress was directly proportional to shear rate for all samples and suggested that the samples were Newtonian fluids. Also, increase in concentration of the fractions led to increase in rate of change of shear stress with respect to shear rate.

Figure 4. Flow curve at 23°C, of different sulfated polysaccharide fractions of from T. turbinata using anion exchange chromatography. A: TtF1; B: TtF2; and C: TtF3.

Figure 5 shows the consistency index of the SPs fractions. Consistency index of TtF2 was highest at all concentrations compared to other two SPs fractions. The result shows that increase in concentration from 0.25–1.00% caused a gradual increase in consistency index for all fractions, while a remarkable increase was observed in TtF1 and TtF2 as concentration increased from 1.00–2.00%. However, further increase in concentration in TtF3 from 1.00–2.00% did not result in an increase in consistency index. Table 4 shows steady shear rheological properties of the SPs fractions. The R² values for all the samples are reasonable high (>0.9) supporting the fitness of Power law model. Increase in concentration of all fractions caused variable increases in viscosity. Fraction TtF2 (0.348 Pa. s) exhibited the highest viscosity followed by TtF1 (0.0152 Pa. s) and TtF3 (0.0026 Pa. s).

Table 4. Steady shear rheological properties of sulfated polysaccharides of fucoidan from T. turbinate.

Sample	Concentration (%)	Flow behavior index, n	Viscosity, η (Pa. s)	Determination coefficient, R^Citation2
TtF1	0.25	0.9717	0.0018	0.9989
	0.50	1.1282	0.0027	0.9962
	1	1.0085	0.0049	0.9997
	2	0.9559	0.0152	0.993
TtF2	0.25	0.9477	0.0025	0.9954
	0.50	0.9508	0.004	0.999
	1	1.0174	0.0086	0.9999
	2	0.9983	0.0348	0.9999
TtF3	0.25	1.0218	0.0014	0.9955
	0.50	1.0512	0.0016	0.934
	1	0.9359	0.0018	0.9991
	2	0.9776	0.0026	0.9993

Figure 5. Consistency index of different fractions sulfated polysaccharide of T. turbinata.

The flow behavior index (n) for all samples at different concentrations varied and lacked a specific relationship with increase in concentration. Flow behavior index ranged from 0.9359 to 1.1282 suggesting that the SP solutions could be a very weak shear thinning material (pseudoplasticity) and most probably Newtonian fluids. The result obtained in this study was comparable with previous reports that stated that SPs, most especially, fucoidan solutions could behave as pseudoplastic or Newtonian material. Rioux et al.^[8] studied the rheological properties of fucoidan extracted from brown seaweeds namely: Ascophyllum nodosum, Fucus vesiculosus, and Saccharina longicruris. It was reported that fucoidan solutions (0.25–1.75%) flow behavior indexes decreased slightly below 1 suggesting that they are Newtonian fluids. Tako^[29] investigated the rheological characteristics of fucoidan isolated from commercially cultured Clasdosiphon okamuranus. Fucoidan solutions at lower concentrations showed shear-thinning behavior at concentrations below 1.5% but plastic behavior at 2.0%. In another report, the flow behavior indexes of fucoidan were low and ranged from 0.7749–0.9266, confirming that fucoidan solutions exhibited pseudoplastic behavior even at higher concentrations (2–15%) of fucoidans.^[30] Likewise, fucoidan pastes at different concentrations (0.1–1.0%) exhibited flow behavior index values in a range of 0.87–1.00, suggesting weak pseudoplastic behavior.^[31]

Rheological properties of a material are the determinant of force of attraction between the molecules of the material and interaction with the medium. Efforts have been made to link chemical properties (molecular weight, polydispersity, branching, sulphate ion content, and uronic acid content) of SPs with their physical rheological properties. Tako^[29] suggested that flow characteristics of fucoidan depends on its intermolecular entanglement in aqueous solution and that random conformation might be adopted in aqueous solution. Our results show that TtF3 with highest molecular weight and high sulfate content but higher polydispers exhibited lowest viscosity.

There are mixed observations with to regard to relationships between fucoidans chemical structure and viscosity. According to Rioux et al.,^[8] low viscosity exhibited by fucoidan extracted from S. longicruris was associated with low molecular weight (44 kDa), smaller proportion of sulfates (18.3%) and uronic acids (5.3%). However, fucoidan of F. vesiculosus that was of lower molecular weight (877 kDa), uronic acid (3.0%), and sulphate ion (19.0%) exhibited higher viscosity compared to fucoidan obtained from A. nodosum with high molecular weight of 1323 kDa, 9.3% uronic acid, and 22.3% sulphate ion. Higher values in uronic acid and sulphate ion of fucoidan obtained from A. nodosum, compared to that of F. vesiculosus, created higher amount of negative charge which lead to extended configuration of the polysaccharide.^[8] This might eventually lead to increase in viscosity of the solution.^[32] Therefore, the speculation of linking chemical structure of fucoidan with rheological property has not been completely achieved. Thus advanced chemical structural analysis like branching and sulphate position is required and might give a reasonable explanation for rheological-chemical linkage of fucoidan.

Thermal degradation of purified SP

The TGA curves of TtF1, TtF2, and TtF3 are shown in Figs. 6a–6c, respectively. Table 5 shows the temperature and weight loss characteristics of the SPs. Three degradation stages were revealed for all the fractions with temperature ranges of 25–200, 200–500, and 500–800, respectively for the first, second, and third stages. Overall, thermal degradation of TtF1 and TtF2 followed the same pattern but differed from that of TtF3.

Table 5. Temperature and weight loss characteristics of the sulfated polysaccharide fractions of T. turbinate.

Fractions	1st stage (25–200°C; moisture loss and pyrolysis)	2nd stage (200–500°C; major devolatilization)				3rd stage (500–800°C; minor devolatilization)		Total weight loss (%)
	WLo (%)	WL1 (%)	WL2 (%)	Ti (°C)	Tp (°C)	WL3 (%)	Tp2 (°C)
TtF1	11.142	33.78	15.54	200	240	13.61	580	75.80
TtF2	9.544	32.58	16.504	200	240	12.10	600	72.28
TtF3	8.202	33.22	9.72	200	260	none	None	54.66

Ti: staring devolatilization temperature; Tp: maximum devolatilization temperature; Te: end of main devolatilization; WL: weight loss.

Figure 6. TGA thermogram of sulfated polysaccharides obtained from T. turbinate. A: TtF1; B:TtF2; and C: TtF3.

The first stage of thermal degradation depicts moisture loss in the samples. The highest moisture loss was observed for TtF1 followed by TtF2 and finally TtF3. Moisture loss of 9.54% observed for TtF2 is similar to the moisture loss of 9.4% reported for laminarin.^[33] The moisture loss of 8.4% in TtF3 varied from moisture content of fucoidans in previous reports. Kim and Lee (2012)^[34] reported 10% moisture content while Anastasakis et al.^[33] reported 12%. This variation might be due to different storage conditions prior to analysis.

The second and third stages of thermal degradation, respectively, represented the major and minor devolatilization processes. The main devolatilization occurs at the same initial temperature (Ti) of 200°C for all samples, but different maximum peak temperature (Tp). A closer value of 195°C has been reported for Ti during thermal degradation of fucoidan;^[35] however, another report stated that the Ti value for fucoidan and laminarin was 175°C.^[33] TtF1 and TtF2 have equal Tp values of 240°C, while that of TtF3 is 260°C. The second stage comprises of 2 phases, percentage weight loss during the phase 1 of all the samples are relatively close values but that of phase 2 differs.

During the third stage of devolatilization process, TtF3 did not show weight loss compared to TtF1 and TtF2 which had 13.61 and 12.10% weight losses, respectively. The total weight losses for TtF1, TtF2, and TtF3 were 75.80, 72.28, and 54.66% respectively. Additional thermal degradation that occurred in the third stage in TtF1 and TtF2 lead to increased total degradation observed in these samples compared to TtF3.

Conclusion

The establishment of structural characteristics of biocompounds is an essential aspect of biochemicals studies because such studies pave the way to insight and understanding of their biological roles at the molecular level. Both chemical and physical properties of SP extract have shown little relationship. Therefore, the difference in chemical and physical properties of the SP from brown seaweed requires further studies.