Purification and bioactivity of fucoidan from Kappaphycus alvarezii Alga

Kappaphycus alvarezii is a type of seaweed containing numerous carbohydrates with biological activities, especially fucoidans. In terms of chemical aspect, fucoidansare fucose polysaccharides (FCSPs) with diverse biological activities including antioxidant, anticoagulant, anti-inflammatory, antiviral, antitumor. Fucoidan is found not only in brown algae, but also in K. alvarezii alga from the central coast of Viet Nam. In this study, fucoidan from K. alvarezii alga (collected at Dam Mon area, Khanh Hoa province) was purified, its structure was characterized, and its biological activities were investigated. The recovery yield of two-stage precipitation by 99 % ethanol was 84.4 %. The protocol of fucoidan purification by ion-exchange chromatography was applied with tris-HCl buffer, elution solvent was NaCl 1M; The yield was 60.99 %. The structure of fucoidan from K. alvarezii from the second fraction was determined by 1H-NMR spectrum with the specific characteristic properties of fucoidan. The antioxidant activity of the purified fucoidan via IC50 was 303.51 ± 0.12 μg/mL (DPPH assay), and 299.97 ± 3.12 μg/mL (ABTS essay). The purified fucoidan exhibited better ability in cases of anticoagulant, antibacterial activities than that of the crude extract. The anti-coagulation time of the purified fucoidan was 22.52 minutes. The minimum inhibitory concentrations (MICs) of the antibacterial activity were 400 μg/mL (Bacillus cereus, Escherichia coli), 500 μg/mL (Aspergillus flavus), and 600 μg/mL (Aspergillus niger).

PURIFICATION AND BIOACTIVITY OF FUCOIDAN FROM

Kappaphycus alvarezii ALGA

Hoang Thi Ngoc Nhon, Le Thi My Ngoc, Nguyen Thi Minh Chi, Nguyen Pham Cam Tien, Nguyen Tuan Kiet, Le Thi Hong Anh*

Food faculty, Ho Chi Minh City University of Food Industry, 140 Le Trong Tan street,

Tay Thanh ward, Ho Chi Minh city

* Email: lethihonganh@gmail.com

Received: 14 August 2019; Accepted for publication: 1 November 2019

Abstract Kappaphycus alvarezii is a type of seaweed containing numerous carbohydrates with

biological activities, especially fucoidans In terms of chemical aspect, fucoidansare fucose

polysaccharides (FCSPs) with diverse biological activities including antioxidant, anticoagulant,

anti-inflammatory, antiviral, antitumor Fucoidan is found not only in brown algae, but also in K

alvarezii alga from the central coast of Viet Nam In this study, fucoidan from K alvarezii alga

(collected at Dam Mon area, Khanh Hoa province) was purified, its structure was characterized,

and its biological activities were investigated The recovery yield of two-stage precipitation by

99 % ethanol was 84.4 % The protocol of fucoidan purification by ion-exchange

chromatography was applied with tris-HCl buffer, elution solvent was NaCl 1M; The yield was

60.99 % The structure of fucoidan from K alvarezii from the second fraction was determined

by 1H-NMR spectrum with the specific characteristic properties of fucoidan The antioxidant

activity of the purified fucoidan via IC50 was 303.51 ± 0.12 μg/mL (DPPH assay), and 299.97 ±

3.12 μg/mL (ABTS essay) The purified fucoidan exhibited better ability in cases of

anti-coagulant, antibacterial activities than that of the crude extract The anti-coagulation time of the

purified fucoidan was 22.52 minutes The minimum inhibitory concentrations (MICs) of the

antibacterial activity were 400 μg/mL (Bacillus cereus, Escherichia coli), 500 μg/mL

(Aspergillus flavus), and 600 μg/mL (Aspergillus niger)

Keywords: bioactivity, fucoidan, ion-exchange chromatography, K alvarezii, purification

Classification numbers: 1.1.1, 1.2.1, 1.4.2

1 INTRODUCTION

Fucoidan is a polysaccharide composed of L-fucose with sulfate ester, found in seaweeds

Fucoidan exhibits a lot of biological activities, such as anticoagulant, antivirus, anti-bacteria,

antifungal, anti-inflammation, and antitumor [1] These activities are related to the chemical

composition of fucoidan, which depends on the structural complexity of fucoidan, algal species,

growth conditions, extraction method, etc [2] The structures of fucoidan can vary, depending

on seasons, the age of the population, species and geographic location [3] Unlike synthetic

antioxidants, fucoidan is a natural substance with significant capacity to avoid or delay radical reactions by forming stable free radicals out of ROS (Reactive oxygen species) [1] Fucose is considered as a key radical scavenger [4] and the ratio between fucose and sulfate content demonstrated the antioxidant activity of fucoidan [5] Regarding the purification, ion-exchange chromatography has been used to purify fucoidan from the extract into fractions, which separates molecules based on the overall charge of the molecule due to its sulfate groups Generally, fucoidan has an overall negative charge [6] A number of reports showed that macroalgae present a broad range of biological activities such as antibacterial, antifungal, antiviral and anti-inflammatory effects Compounds with cytostatic, antiviral, anti-helminthic, and antifungal activities have been detected in green, brown, red algae and other aquatic weeds

[7] K alvarezii, a red alga, contains mainly carbohydrates, proteins, minerals, pigments

Carbohydrates (50 - 60 %) usually concentrate at the cell walls, including cellulose and sugars with biological activity [8] In this study, 1 H-NMR spectrum and several biological activities of the fucoidan from this alga are reported

2 MATERIALS AND METHODS 2.1 Materials

Fresh K alvarezii alga was collected in Dam Mon area (Khanh Hoa province, Vietnam)

Salt, sand, and epiphytes were removed with tap water The samples were then rinsed carefully with fresh water and stored in a plastic bag at -5 C To prepare for the experiment, the alga was dried to about 10 % moisture, ground and sieved to one mm inhomogeneous size

2.2 Methods

2.2.1 Sample preparation

Extraction: The milled alga (120 g) was immersed in 85% ethanol under mechanical stirring at room temperature for 12 hours The final residue was extracted with HCl (pH 2) at 90 o

C for 3.5 hours The extract was then precipitated with TCA (trichloroacetic acid) at a temperature of 4 oC in 1 hour, then centrifuged (5500 rpm, 15 minutes) to remove proteins and gained the extract

Pre-purification: The extract was precipitated with 99 % ethanol (ethanol:extract 36:69 w/w) yielding an extract with 30 % alcohol The extract was left standing for 12 hours at 4 oC, then centrifuged to remove the precipitate Then, 99 % ethanol was added to the supernatant (ethanol: extract 40:29 w/w) yielding a solution with 70 % alcohol, kept at 4 oC for 12 hours, then centrifuged to collect the precipitated fucoidan [9]

2.2.2 Fucoidan purification by ion-exchange chromatography

Fucoidan precipitate (1 g) was dissolved in 5 mL of distilled water and applied to a column (1.2×12 cm) of DEAE-cellulose, pre-equilibrated with water, with a low rate of 1 mL/minute This was continued until the pH of equilibration of water and stepwise elution with distilled water by passing NaCl in increasing concentrations (0.5 M, 1 M, 1.5 M, 2 M) with a flow rate of 0.5 mL/min Three types of buffers (Tris-HCl, phosphate, acetate) were investigated Fractions were collected and fucoidan content and purity were determined

2.3 Analytical methods

2.3.1 Fucoidan determination by spectrophotometer [10]

Standard curve setting: Standard fucoidan (Sigma) was used as the standard Three

replicates of the standard were prepared in different concentrations with distilled water ranging from 10-100 µg/ml Sample solution of each concentration (1.0 mL) was added to a standard test tube The test tubes were cooled on ice at 4 oC (2-3 minutes), 4.5 mL of sulfuric acid (85 %) is added and the samples are homogenized with the help of glass stirrer Tubes were then cooled under running tap water, and 0.3 % cysteine hydrochloric acid was added to the tubes and mixed Tubes were placed in total darkness for 2 hours, then the absorbance was measured on a spectrophotometer at 396 nm and 430 nm A blank with distilled water treated under the same manner was used for the zero Absorbance = (A396 nm – A427 nm)

Determination of fucoidan content: The same above protocol was used for fucoidan

determination from the samples, in which the sample is replaced by standard fucoidan

2.3.2 Determination of fucoidan structure by nuclear magnetic resonance (NMR) spectra [11]

The application of NMR spectroscopy would also be very useful to obtain more structural information on fucoidan by identifying the present residues and how this polysaccharide is linked together The side chains can also be determined and should lead to a much better understanding of the various biological properties that fucoidan have The spectra were obtained

on a spectrometer provided with a 5 mm probe at room temperature The solution of polysaccharide samples in H2O was sonicated at 20 kHz, and then D2O was added to produce a solution containing 40 mg of calciumin 0.4 mL of 1:1 H2O – D2O Acetone was added as an internal standard (referred to Me4Si by calibrating the acetone methyl group to 31.1 ppm) Typical parameters were as follows: maximum acquisition time, no relaxation delay, 90°-pulse angle, and 40,000 scans

2.3.3 Antioxidant activity

DPPH method [2]: Free radical scavenging activity of fucoidan extract was measured by 1,1- diphenyl-2-picryl hydrazyl (DPPH) In brief, a 0.1 mM solution of DPPH in ethanol was prepared This solution (1 mL) was added to 3 mL of different extracts in ethanol at different concentrations (5, 10, 15, 20, 25, 30 µg/mL) Here, only those extracts are used which are solubilized in ethanol and their various concentrations were prepared by the dilution method The mixture was shaken vigorously and allowed to stand at room temperature for 30 minutes then, measured at 517 nm by using a spectrophotometer

ABTS method [12]: Cationic ABTS is a blue free-radical at 734 nm When an antioxidant substance is added to the solution containing ABTS+, it will be deoxidized into ABTS; 0.1 mL of sample was mixed with 1.0 mL of ABTS solution Samples were diluted at concentrations of

625, 1250, 2500, 5000, and 10000 μg/mL Absorbance at 734 nm was measured to determine antioxidant activity in comparison with the Trolox standard in an ABTS persulfate medium for 2 days at room temperature in the dark

2.3.4 Anticoagulant activity [13]

The anticoagulant activity of fucoidan was determined by the United States pharmacopia

method: 0.8 mL fucoidan solutions (the extract, the purified fucoidan) at different concentrations

(600, 500, 400 g/mL), 0.8 mL sodium heparin standard solvent (0.7 USP/0.8 mL) and 0.8 mL

NaCl 9 % were added in successive order to the samples 1 mL of plasma and 0.2 mL CaCl2 (1 %) were added prior to mixing the solution in each tube The clotting time of the blood was

recorded

2.4 Data analysis

Each experiment was carried out in triplicate The results were presented as a mean ± error

value Data were analyzed by an analysis of variance (p < 0.05), the means separated by

Duncan’s multiple range tests The results were processed using Office Excel 2013 and JMP

software

3 RESULTS AND DISCUSSION 3.1 Fucoidan purification by ion-exchange chromatography

Fucoidan precipitation protocol was done according to section 2.2.2 The content and the

purity of fucoidan contained in the two-stage precipitate method were 191.378 g/mL and 19.47 %, respectively Therecovery yield was 84.40 % This method was applied by T

Marudhupandi et al [9] to pre-purify fucoidan from seaweed The DEAE ion-exchange

chromatography was carried out with 3 buffers of Tris-HCl, phosphate, acetate and four given

NaCl concentrations The effects of buffers and concentrations of NaCl were depicted in Figure 1

Figure 1 The effects of buffers (a) and concentrations of NaCl elution (b)

The efectiveness of three types of buffers used in ion-exchange chromatography were

statistically significantly different The Tris-HCl buffer had the better result (623.56 μg/mL) than

that of phosphate buffer (540.68 μg/mL) and acetate buffer (473.56 μg/mL) (Figure 1.a) Mak

W et al [14] purified fucoidan by ion-exchange chromatography using Tris-HCl buffer pH 7.4,

and achieved the highest purity of 69.98 % In this study, Tris-HCl buffer pH 7 was selected

Figure 1b showed that the fucoidan content in the fraction 1 M was the highest (518.303 μg/mL)

and continued to decrease in the subsequent fractions A possible explanation was that the

elution of NaCl 0.5 M was not strong enough in terms of ionic force, while NaCl 1 M was strong

enough to elute fucoidan attached on DEAE However, the subsequent concentrations of

stronger ionic strength other impurities also eluted Mak W et al used saline for the stage of

elution in fucoidan purification by ion-exchange chromatography The results showed that the concentrations of saline 1 M and 1.5 M were suitable for the elution of fucoidan [14] To purify

fucoidan, Isnansetyo A et al also eluted with distilled water and gradient 0.5 to 3 M NaCl [15], Kim W J et al eluted with water containing increasing concentrations of NaCl (0.5, 1.0, 1.5,

2.0, 2.5, 3.0 M), until no more carbohydrate was detected [16] In this study, the elution of the NaCl concentration of 1 M was chosen From above results, ion-exchange chromatography was carried out with the chosen parameters, and the results were shown in Table 1 The second fraction was highest in terms of fucoidan content 630.16 (μg/mL) and purity (61.14 %), followed

by fractions 3; 4; 5 The subsequent fractions experienced a gradually decreasing trend At NaCl 0.5 M, the elution was done several times to remove impurities Therefore, the purity of the fucoidan fraction (obtained from elution with NaCl 1 M) was high The content and purity of fucoidan in subsequent fractions decreased gradually The last fraction contained mainly salt

Table 1 The fucoidan content of post-ion exchange chromatography with Tris-HCl buffer

Fractions Content

(µg/mL)

Purity (%) Fractions

Content (µg/mL)

Purity (%) Fractions

Content (µg/mL)

Purity (%)

3.2 1 H-NMR spectroscopy

The 1H-NMR spectrum of fucoidan with different molecular weights is very complex, depending on algae species or extract conditions The 1H-NMR spectra of fucoidan collected from the second fraction at the elution of 1M saline are illustrated in Figure 2 The 1H-NMR spectrum contained several intense signals in the a-anomeric (5.0–5.6 ppm) and high-field (1.2– 1.5 ppm) regions of the α-L-Fucopyranose ring [17] The signals of the last region at 1.32 and 1.43 ppm were assigned to C6 methyl protons of L-fucopyranose; several intense and narrow signals at 2.2 ppm arise from CH3 protons of O-acetyl groups [12, 18] In the 1H-NMR spectrum

of fucoidan also contained strong signals in the proton (H6) high-field of the methyl group and signals at 5.0-5.1 ppm, characteristic of the anomeric (H1) proton ring of the ring α-L-Fucpyranose linkage 1→3 [18] In addition, 1H-NMR spectra had signals of 3.3 ppm and 5.3 ppm, which was characterized by the H6 and H1 protons of the β- D-Galactose Finally, the signal at 2.1 ppm indicated the presence of the O-acetyl group Based on these results, the structure was characterized as fucoidan

3.3 Antioxidant activity

As the concentration increased, the DPPH radical elimination capacity increased The antioxidant activity of fucoidan from fraction 2 (purified by DEAE-cellulose) is shown in Table

2 To assess the antioxidant activity, the value of IC50 has been used The IC50 value of purified

fucoidan from K alvarezii alga was 299.97 μg/mL Wang et al [13] reported that the capacity of

free radical scavenging DPPH of fucoidan depends on fucoidan content Both the sulfate content and the high molecular weight of fucoidan are necessary for determining the biological activity

of fucoidan According to Skriptsova et al [19], other factors such as the location of the sulfate

group, the level of monosaccharides also affected the biological activity of fucoidan The study

of W Maka [13] showed that fucoidan extracted from brown algae U Pinnatifida at 1000

μg/mL captured free radicals at 86.60

Figure 2 1H-NMR spectra of fraction 2

Table 2 Antioxidant activity of fucoidan (DPPH, ABTS)

Concentration (µg/mL) DPPH method ABTS method

IC50 IC50 = 303.51 ± 1.65(µg/mL) IC50 = 299.97 + 1.75(µg/mL)

3.4 Antibacterial activity

The zone of inhibition of the fucoidan extract and purified fucoidan (fraction 2) were shown in Table 3 The fucoidan extract was concentrated to gain higher concentrations, and the purified fucoidan was diluted to lower concentrations from 300 to 600 μg/mL

Table 3 showed that from 400 μg/mL, both the fucoidan extract and purified fucoidan could

inhibit E coli and B cereus The maximum inhibition zones were recorded at 600 μg/mL with

7.40 - 8.63 mm for the extract and 9.03 - 10.50 mm for the purified fucoidan The purified fucoidan had better antibacterial activity than that of fucoidan extracts The extract and the

purified fucoidan had more effectiveness on B cereus than E coli It can be observed that

gram-negative bacteria were more susceptible than that of gram-positive species The antibacterial property of the plants appears to have justified their use for the treatment of wounds, which are contaminated through bacterial infection [20] The results of Ming Liu’s study [21] showed that

fucoidan from L japonica strongly inhibited the growth of gram-negative bacteria (E coli, S

aureus) It was also reported that the crude fucoidan extract from S wightii contained substances

that inhibited the growth of bacteria causing canker in citrus and blight in paddy [22]

Table 3 The zone of inhibition of the fucoidan extract and purified fucoidan

Concentratio

ns (µg/mL)

The zone of inhibition

(mm)

Concentrations (µg/mL)

The zone of inhibition

(mm) Fucoidan extract Purified fucoidan

3.5 Antifungal activity

The antifungal activity of the 4 dilutions of purified fucoidan (fraction 2) was performed on

Petri dishes The tested molds were A niger and A flavus The diameter of the zone of inhibition

was shown in Table 4

The purified fucoidan could inhibit the growth of A niger and A flavus The MIC value and the zone of inhibition of A niger and A flavus were 600 μg/mL (11.17 mm) and 500 μg/mL

(9 mm), respectively The activity of purified fucoidan was better than that of the extract This may be explained by the fact that the impurities in the extract may impede the diffusion of fucoidan into the agar, leading to a decrease in the resistance of the extract to the molds

Table 4 The zone of inhibition of the fucoidan extract and purified fucoidan

Concentrations

(µg/mL)

The zone of inhibition

(mm)

Concentrations (µg/mL)

The zone of inhibition

(mm)

3.5 Anticoagulant activity

Anticoagulant times of the extract, purified fucoidan and positive controls (heparin, NaCl) were presented in Table 5

Table 5 The anticoagulant activity of the extract and purification fucoidan

Crude extract 10.27 ± 0.46 Purified fucoidan 22.52 ± 1.17

The average time for coagulation of fucoidan extract (10.27 minutes) and purified fucoidan

(22.52 minutes) were higher than that of NaCl solution (0.9 %) (7.31 minutes) However, it was

lower than that of heparin (28.31 minutes) The anticoagulation times increased with the increase

of fucoidan concentrations The anticoagulant activity of fucoidan is attributed to the sulfate

content and molecular weight of fucoidan [1] A higher sulfate content results in better

anticoagulant activity [16] The anticoagulant activity of fucoidan isolated from E kurome, H

fusiforme, and L angustata var Longissima was higher than that of heparin The position of the

sulfate groups on the sugar substrates was also important for the fucoidan coagulant activity The

sulfate group of fucoidan at C-2 or C-3 illustrated its anticoagulant activity, whereas sulfate at

C-4 did not show this activity Fucose and galactose also have an effect on anticoagulant activity

[22] The uronic acid component did not directly affect anticoagulant activity, but it indirectly

increased the anticoagulant activity of fucoidan by making the structure more flexible [1]

Fucoidan has a great potential for use as an anticoagulant, antithrombotic or functional food, and

medicinal product

4 CONCLUSIONS

Fucoidan was purified by ion-exchange chromatography with tris-HCl buffer, elution

solvent NaCl 1M; The yield was 60.99 % The structure of fucoidan via 1H-NMN spectrum has

the specific characteristic properties of fucoidan Fucoidan from K alvarezii also exhibited

biological properties such as antibacterial, antifungal and anticoagulant activities The fraction 2

at the elution of NaCl 1 M showed higher amount and bioactivity than other fractions

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