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Characterization of alginic acid extracted from Sargassum wightii and determination of its anti-viral activity of shrimp Penaeus monodon post larvae against white spot syndrome virus

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RESEARCH ARTICLE
CHARACTERIZATION OF ALGINIC ACID EXTRACTED FROM SARGASSUM WIGHTII
AND DETERMINATION OF ITS ANTIVIRAL ACTIVITY ON SHRIMP PENAEUS
MONODON POSTLARVAE AGAINST WHITE SPOT SYNDROME VIRUS
*1Sivagnanavelmurugan, M., 2Radhakrishnan, S., 3Palavesam, A., 1Arul, V. and 3Immanuel, G.
1Department of Biotechnology, Scholl of Life Sciences, Pondicherry University, Pondicherry. India- 605014.
2Central Electrochemical Research Institute, Karaikudi, India
3Marine biotechnology Division, Centre for Marine Science and Technology, Manonmaniam Sundaranar
University, Rajakkamangalam – 629 502, Kanyakumari District, Tamilnadu, India
Received 18th February, 2018; Accepted 16th March, 2018; Published 30th April, 2018
ABSTRACT
The polysaccharide- alginic acid was extracted from brown seaweed Sargassum wightii and the yield observed was 3.932%. The
purity of alginic acid was determined by preliminary phytochemical analysis and the result indicated that it has only the presence
of carbohydrates and its derivative saponins. The physicochemical properties of alginic acid were analyzed. The FT-IR, 13C and
1H NMR analysis indicated the presence of carbons and anomeric protons of guluronic acid and manuronic acid in purified and
hydrolyzed alginic acid, respectively. The characterized alginic acid was enriched with instar II stage Artemia nauplii at 100, 200,
300 and 400 mg L-1 concentrations for 12 h and they were fed to Penaeus monodon postlarvae (PL15) for 20 days. After feeding
experiment, the P. monodon PL35 were challenged with WSSV. The control group of shrimp PL fed with unenriched Artemia
nauplii showed 100% mortality within 8 days, but the alginic acid (100-400 mg L-1) enriched Artemia nauplii fed shrimp PL
showed less mortality (79 100 %) within 21 days of WSSV post challenge. The reduction in mortality percentage of alginic
acid enriched Artemia nauplii fed groups over control group was ranged between 17.15 and 49.99 %. The RT-PCR analysis
confirmed the considerable reduction of WSSV DNA copy numbers (71757 – 5.73 WSSV DNA copies) in shrimp postlarvae
with respect to the concentration of alginic acid. The present result concluded that P. monodon PL fed with alginic acid of S.
wightii enriched Artemia nauplii has increased the resistance against WSSV infection.
Key words: Alginic acid; Sargassum wightii; Penaeus monodon; WSSV; Artemia franciscana.
Copyright © 2018, Sivagnanavelmuruga et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Citation: Sivagnanavelmurugan, M., Radhakrishnan, S., Palavesam, A., Arul, V. and Immanuel, G., 2018. “Characterization of alginic acid
extracted from sargassum wightii and determination of its antiviral activity on shrimp penaeus monodon postlarvae against white spot syndrome
virus” International Journal of Current Research in Life Sciences, 7, (04), 1863-1872.
INTRODUCTION
Alginates are one of the polysaccharides naturally present in
the cell walls of brown seaweeds (Kloareg and Quatrano,
1988). These polysaccharides show interesting rheological
properties: they enable to enhance aqueous solutions viscosity
at low concentration, and to form gels or thin films. They are
widely used in various fields of industries such as textile, food,
paper, cosmetics, pharmaceuticals, etc. (Perez et al., 1992).
Alginate has a combined feature of abundant resources with a
linear copolymers of L-guluronic acid and D-mannurunic acid
units (Xu et al., 2006). The major structural polysaccharide of
brown seaweeds in alginic acid, a linear copolymer of (1→4)
linked β-D-mannopyranuronic acid (m) and (1→4) linked α-L-
*Corresponding author: Sivagnanavelmurugan, M.,
Department of Biotechnology, Scholl of Life Sciences, Pondicherry
University, Pondicherry. India- 605014.
gulopyramuronic acid (G) residues, arranged in
heteropolymeric and homo polymeric blocks (Painter, 1983;
Larsen et al., 2003). The content of uronic acids with species
and tissue types, and partial hydrolysis of alginic acid allows
the preparation of fractions enriched in water and
homopolymeric blocks (Haug et al., 1974; Craigie et al.,
1984). The sulfated polysaccharides (SPs) have been shown to
possess antiviral activities. It has been reported that high
molecular weight sulfated galactans (SG) from red seaweeds
have antiviral properties against herpes simplex virus (HSV),
human cytomegalo virus (HCMV), dengue virus (DENV) and
respiratory syncytial virus (RSV) (Mazuder et al., 2002; Hidari
et al., 2008). Hidari et al. (2008) reported that fucoidan from
the brown marine alga Cladosiphon okamuranus inhibits
DEN2 infection. White spot syndrome virus (WSSV) is a
pathogen that has devastated the shrimp farming industry
(Lightner and Redman, 1998; Jiang et al., 2006); currently it is
considered as the most serious shrimp viral pathogen in the
ISSN: 2319-9490
International Journal of Current Research in Life Sciences
Vol. 07, No. 04, pp.1863-1872, April, 2018
Available online at http://www.ijcrls.com
world (Flegel, 2006; Sanchez-Martinez et al., 2007). WSSV is
found in almost all shrimp producing countries and lethal to all
commercially cultivated penaeid shrimp species (Wang et al.,
2000; Sanchez-Martinez et al., 2007; Escobedo-Bonilla et al.,
2008). WSSV is a rod-shaped enveloped dsDNA virus
(275×120 nm in size) with a tail-like appendage at one end.
WSSV is identified by the presence of white spot on the inner
surface of the exoskeleton of shrimp from which the name is
derived (Lo et al., 1996). Other clinical signs include anorexia,
lethargy and reddish discoloration of the body (Wang et al.,
1999). Cumulative mortalities in infected populations may
reach 100% within 2-10 days of the onset of clinical signs
(Chou et al., 1995; Lightner, 1996; Xu et al., 2006). Previous
strategies generally used to control WSSV including
immunostimulation, neutralization, vaccination, quarantining,
and environmental management (Xiang, 2001). Protective
effect of various herbal immunostimulants has been reported
against WSSV infection in shrimp P. monodon
(Rameshthangam and Ramasamy, 2007; Balasubramanian et
al., 2007). The antiviral plant extract of Cynodon dactylon
against WSSV infection and immunostimulatory effect on P.
monodon through oral administration was studied
(Balasubramanian et al., 2008).
Administration of cidofovir ((S)-1-3-hydroxy-2-phosphonyl
methoxy propyl cytosine) (HPMPC) supplemented with a
marine blue green algae Spirulina platensis in shrimp diet has
been shown to delay the mortality of WSSV infected shrimp
(Rahman et al., 2006). In vivo screening of mangrove plants
for antiWSSV activity in P. monodon and evaluation of
Cereops tagal as a potential source of antiviral molecules has
been reported by Sudheer et al. (2011). Huynh et al. (2011)
have reported the white shrimp L. vannamei immersed in
seawater containing S. hemiphyllum var. chinense powder and
its extract showed increased resistance against WSSV.
Immanuel et al., (2012a) and Sivagnanavelmurugan et al.,
(2012) have reported the effect of fucoidan from brown
seaweed S. wightii on WSSV resistance and immune activity in
shrimp P. monodon. Immanuel et al., (2012b) have studied the
effect of sodium alginate extracted from S. wightii retards
mortality in WSSV challenged shrimp P. monodon. Ergosan,
an algal extract containing alginic acid was also observed to
increase the nonspecific defense response of snake head
Channa striata (Miles et al., 2001), rainbow trout
Oncorhynchus mykiss (Peddie et al., 2002) and sea bass
Dicentrarchus labrose (Bagni et al., 2005). Considering the
importance of the above, the present study was undertaken to
extract and characterize the alginic acid from brown seaweed
S. wightii and to determine its antiviral effect on P. monodon
post larvae against WSSV.
MATERIALS AND METHODS
Collection of seaweed
The brown seaweed S. wightii was collected from the coastal
villages of Kanyakumari District, Tamilnadu, India. The
collected seaweed was washed thoroughly and dried under
shade at room temperature. The dried seaweed was ground
well by using mixer grinder and sieved using nylon sieve (0.45
µm pore size) in order to remove unpowdered materials
(Immanuel et al., 2010).
Extraction of alginic acid
The alginic acid was extracted from the brown seaweed S.
wightii by following the modified method of Torres et al.
(2007). 100 g of milled seaweed sample was weighed and
soaked in 2% formaldehyde in an air tight conical flask for 24
h. After 24 h, the formaldehyde solution was filtered out and
the residue was washed with distilled water for 2 to 3 times.
Then 0.2 M HCl was added to the residue and kept at room
temperature for 24 h. After 24 h, the solution was removed and
the residue was washed with distilled water for 2 to 3 times.
The residue was extracted with 2 % sodium carbonate for
overnight and the extract was filtered through muslin cloth
bag. Then 5 % HCl was added to the filtrate for precipitation
of alginic acid. The precipitate was separated by centrifugation
method (5000 rpm for 15 min). Further the product was dried
and made in to powder and calculate the yield.
Determination of purity of alginic acid (phytochemical
analysis)
To determine the purity of alginic acid, tests for alkaloids,
carbohydrates, flavonoids, steroids, terpins, saponins, tannins
and phenols were carried out by the methods proposed by
Harborne (1973); Trease & Evans (1989) and Sofowora
(1993).
Physicochemical properties of alginic acid
The colour, odour, taste and texture of extracted alginic acid
were evaluated by using the methodologies described
previously by Kumar et al., (2011). A digital pH meter (Model
2001, Digisum Electronics System) was used to determine the
pH of 1% alginic acid solution. Moisture content of the powder
was determined using the Indian Standards Institution method
(ISI 1984). Protein, carbohydrate, lipid, fucose and sulfate
contents of alginic acid were estimated using standard methods
(Lowry et al., 1951, Seifter et al., 1950, Folch et al., 1957,
Dubois et al., 1956, Dodgson and Price, 1962). Ash content
was determined by combusting 1 g of alginic acid powder in a
silica crucible in a muffle furnace at 600°C and, once cooled
and the weight of ash was determined. The prepared ash was
then boiled in 25 ml of 2 N HCl for 5 min, and any insoluble
ash was collected on ash-free filter paper and washed with hot
water. This insoluble ash was transferred into a silica crucible,
combusted and weighed as described above. The procedure
was repeated to get an average weight to accurately determine
the percentage of acid-insoluble ash. Ash was boiled similarly
in 25 ml water for 5 min, and insoluble ash was collected and
washed as above, transferred to a silica crucible, combusted for
15 min and weighed. The procedure was repeated to get an
average weight of water-insoluble matter that was subtracted
from total ash weights to determine the percentage water-
soluble ash.
Hydrolysis of alginic acid
In order to reduce the viscosity of the sample as well as to
convert the polysaccharide into monosaccharide, the purified
alginic acid was subjected for hydrolysis. For this, 20mg of
alginic acid was dissolved in 5ml of distilled water and heated
at 90o C for 1h. Then 1ml of 0.1 N HCl was added to the
sample, it was heated at 90o C for 2h and further the sample
was freeze dried (Marais and Joseleau, 2001).
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1863-1872, April, 2018
FT-IR analysis
The qualitative analysis of the active principles of hydrolyzed
alginic acid was done by Fourier Transmission Infra Red
(FTIR) method, described by Kemp (1991).
13C and 1H NMR analysis
After hydrolysis, the alginic acid sample was dissolved in
0.5ml D2O (Deutrium dioxide), and the proton number and
carbon number of alginic acid were identified and confirmed
by 1H and 13C NMR experiments using a Bruker Biospin
Avance 400 NMR spectrometer (1H frequency = 400.13 MHz,
13C frequency = 100.62 MHz) at 298 K using 5-mm broad
band inverse probe head equipped with shielded z-gradient and
XWIN-NMR software version 3.5 using TMS as an internal
reference. One-dimensional 1H and 13C spectra were obtained
using one pulse sequence. One-dimensional 13 C spectra using
Spin Echo Fourier Transform (SEFT) and Quaternary Carbon
Detection (QCD) 42 sequences were also performed to aid the
structure identification (Jayaprakash and Kalaiselvi, 2007).
Artemia enrichment
To determine the experimental treatment concentrations, the II
instar stage of Artemia franciscana nauplii (Great Salt Lake,
USA) were fed (enriched) with alginic acid. For this, four
different concentrations viz 100, 200, 300 and 400 mg L-1 of
alginic acid were prepared individually as enrichment media.
The Artemia nauplii were stocked at the rate of 20 ml-1 in total
volume of 5 L seawater in glass containers. Mild aeration was
given into the medium in order to maintain the oxygen level
and to keep uniform dispersion of the dietary particles of
alginic acid in the medium. The enrichment media were
delivered into two doses at 6 h intervals to the Artemia nauplii,
the enrichment duration was 12 h. To ensure the encapsulation
of the diets, the enriched Artemia nauplii were examined under
the microscope to assess 100 % gut loading. After 12 h, the
encapsulated Artemia nauplii were sieved from the respective
containers, washed carefully and kept individually ready for
feeding.
Collection and maintenance of experimental animal
The shrimp P. monodon postlarvae (PL7) were obtained from
Matsyafed. Hatchery (Quilon, Kerala). Immediately after
arrival in to the laboratory, the PL were stocked in 1000 L
fibre glass tank at room temperature (28 1º C) with the
salinity of 32 1 ppt. Natural filtered seawater was used and it
was well aerated to maintain the oxygen level above 6 ppm.
The PL were kept in the tank for 8 days and fed with live feed
(unenriched Artemia nauplii) for acclimatization prior to start
of the experiment.
Feeding experiment
After measuring the length and weight, uniform (0.0108
0.0036 g) size of P. monodon postlarvae at PL15 stage were
selected from the acclimatized stock and transferred in to
individual experimental tanks (control-unenriched Artemia
diet; alginic acid with respective concentrations of 100, 200,
300 and 400mg L-1 enriched Artemia diet), each containing
100 L of filtered sea water at ambient temperature (28 ± 10C)
and salinity (32 1 ppt). The PL were maintained at the
stocking density of 5no’s L-1.
Mild aeration was given continuously in order to maintain the
optimal oxygen level. An ad libitum feeding regime was
applied to all tanks throughout the experiment, and the food
(enriched Artemia nauplii) density was adjusted 3 times a day
(6:00, 14:00 and 18:00 h) at the rate of 30, 30, and 40 %,
respectively. The control group was fed with unenriched
Artemia nauplii. The uneaten Artemia nauplii were collected
after the respective hours of feeding and 50% water was
exchanged daily during the experimental period. To maintain
the nutritional quality of Artemia, the remaining enriched
Artemia nauplii were kept in cold storage at 4 to 100C with
gentle aeration (Leger et al., 1983). The experiment was
prolonged for 20 days (PL15–35). Simultaneously, triplicate
tanks were maintained in each group.
Preparation of viral inoculum
The WSSV-infected P. monodon with prominent white spots
on the exoskeleton were collected from local shrimp farms.
Head soft tissue from cephalothorax including gills was
homogenized and centrifuged at 3000 xg for 20 min at 4oC.
The supernatant was recentrifuged at 8000 xg for 30 min at
4oC and the final supernatant was filtered through a 0.4-m
membrane filter. The filtrate was then stored at -20oC for
infectivity studies (Yoganandhan et al., 2003). The presence of
WSSV in inoculum was checked by nested PCR and this result
showed severe infection (912bp) equal to 2000 WSSV DNA
copies (IQ 2000TM manual).
WSSV challenge experiment
After feeding experiment, the pathogenesity of WSSV to P.
monodon PL 35 was carried out separately by immersion
(bath) challenge at 2 animals per litre (100 PL 50 L-l)
respectively, were reared separately in FRP tanks (75 L)
containing 50 L of sterilized, aerated sea water. Air stones and
air tubes were sterilized by immersing in 2.6 % sodium
hypochlorite and then washed thoroughly with sterilized tap
water before use. The tanks were covered to prevent
contamination. Aseptic techniques were observed throughout
the experiment. The PL were fed on the respective enriched
Artemia nauplii diet and control group was fed with
unenriched Artemia nauplii. The WSSV inoculum (stored fluid
filtrate) was added to the tank water at a volume equal to 0.1%
of the total rearing medium (1 ml L-1) (Chen et al., 2000).
Simultaneously, triplicate tanks were maintained in each
group. After inoculation of WSSV, the survival of PL was
monitored at regular intervals of 8 h until all the animals had
succumbed. PL not reacting to gentle mechanical stimulation
with a soft paintbrush was considered to be dead. The non-
reacting animals were removed from the respective tanks
during each observation intervals. The results obtained in
every 8 h intervals were pooled and presented as per day
interval. The challenge experiment was conducted for 21 days.
The cumulative mortality index (CMI) was calculated by the
formula described in Immanuel et al. (2012b).
Real Time Polymerase chain reaction (RT-PCR) analysis
After challenge experiment, the WSSV infection in P.
monodon larvae was detected by RT-PCR analysis. The
experimentally infected shrimp PL were preserved in 70%
ethanol and subsequently were rehydrated in distilled water for
1h before the RT-PCR analysis.
1865 International Journal of Current Research in Life Sciences, Vol. 07, No. 04, pp.
1863-1872, April, 2018
The RT-PCR analysis for WSSV DNA quantification was
performed by the methodology described in Immanuel et al.
(2012a).
Statistical analysis
The data obtained in the present study were expressed as Mean
± SD and were analyzed using one way ANOVA at 5%
significant level. Further a multiple comparison by Tukey’s
test was conducted to compare the significant differences
among the parameters using computer software Statistica 6.0
(Statosoft, UK).
RESULTS AND DISCUSSION
Yield of alginic acid
The yield of alginic acid obtained from S. wightii was 3.932 ±
0.22 %. Generally, the alginate content of various seaweeds is
varied much. Torres et al. (2007) reported the yield of alginate
as 16.90% extracted from S. vulgare. Similarly, Davis et al.,
(2004) found the yield within the range of 21.1 24.5 % in S.
fluitans and 16.3 – 20.5% in S. oligocystum with variations
being depended on the methodology followed for alginate
extraction, as well as species which are used for extraction. In
the present study, the yield of alginic acid extracted from
brown seaweed S. wightii was very low with 3.932 ± 0.22 %.
Larsen et al. (2003) reported that, a change in yield of alginate
in different seaweeds like S. dentifolium (3.25 %), S.
asperifolium (12.4 %) and S. latifolium (17.7 %) was due to
species dependent variation and also extraction methods
followed.
Purity of alginic acid
The purity of alginic acid was determined through
phytochemical tests. The result indicated that, the
phytochemical constituents such as alkaloids, saponins,
tannins, phlobatannins, flavonoids, steroids, terpenoids, cardiac
glycosides and phenols were absent.
Only carbohydrate as well as sugar derivative of saponins were
found to be present in alginic acid, which confirmed the better
purity of the algnic acid (Table 1). Similarly, Kumar et al.
(2011) have determined the phytochemical characteristics as
well as the purity of tamarind seed polysaccharide, which
indicated the absence of alkaloids, steroids, flavonoids,
saponins, tannins and phenols, however only carbohydrate was
found to be present, which confirms the purity of this
particular polysaccharide.
Physicochemical characters of alginic acid
The organoleptic characters such as colour, odour, taste and
texture of alginic acid were in the order of white to yellowish
brown colour, odourless, salty taste and powdery appearance,
respectively. The pH of 1% alginic acid solution was 2.35. The
moisture content of alginic acid observed was 18 ± 1.07%.
Similarly, Cyber colloids Ltd. (E400 Alginic acid) have
reported the color, odour, texture, pH, moisture content of
alginic acid were white to yellowish brown color, nearly
odourless, granular or powder, 2 to 3 and 15%, respectively.
The Good Scent Company, have studied the physical
parameters of alginic acid of seaweed and they observed the
color, odour, taste and texture of alginic acid were white to
pale yellow color, odourless and tasteless, respectively.
Chemical Book. (2008) have published the colour of alginic
acid of seaweed was off white to light yellow powder. FMC
BioPolymer (2008) have reported that the color, odour, texture,
taste and pH of alginic acid were white to yellowish color,
odourless, tasteless, free flowing powder, 1.5 - 3.5 (in 3%
aqueous dispersion), respectively. The major biochemical
component of alginic acid was carbohydrate (44.36 ± 1.42%)
with little amount of protein (5.82 ± 0.72) and lipid (4.06 ±
0.341%) contents. The fucose content of alginic acid was 28.99
± 1.09%. The ash values such as total ash, acid insoluble ash
and water soluble ash of alginic acid were 1.41 ± 0.032, 0.098
± 0.0064 and 0.676 ± 0.052%, respectively. The sulphate
content of alginic acid was 25.91 ± 0.390 % (Table 2).
Table 1. Determination of purity of alginic acid extracted from brown seaweed S. wightii through phytochemical analysis3
Phytochemical tests Phytochemical characteristics
Alkaloids -
Saponins +
Tanins -
Phlobatannins -
Flavonoids -
Steroids -
Terpenoids -
Cardiac glycosides -
Phenols -
Carbohydrates +
+ : Present ; - : Absent
Table 2. Physicochemical and organoleptic characters of alginic acid extracted from the brown seaweed S. wightii
Characters Parameters Alginic acid
Organoleptic
characters
Colour White to yellowish brown colour
Odour Odourless
Taste Salty taste
Physical
characters
Texture Powder
pH 2.35
Moisture content (%) 18.0 ± 1.07
Chemical
characters
Protein (%) 5.82 ± 0.72
Carbohydrate (%) 44.36 ± 1.42
Lipid (%) 4.06 ± 0.341
Fucose (%) 28.99 ±1.09
Ash content (%) 1.41 ± 0.032
(i) Acid insoluble ash (%) 0.098 ± 0.0064
(ii) Water soluble ash (%) 0.676 ± 0.052
Sulphate content (%) 25.91 ± 0.390
1866 International Journal of Current Research in Life Sciences, Vol. 07, No. 04, pp.
1863-1872, April, 2018
Similarly, Torres et al. (2007) have studied the biochemical
composition of alginate extracted from S. vulgare. They
reported that the protein values determined were 1.1 and 1.0
for S. vulgare low-viscosity alginate (SVLV) and S. vulgare
high-viscosity alginate (SVHV) samples, respectively. The
moisture and ash contents of SVLV and SVHV were 14 &
16% and 2 & 1%, respectively. Larsen et al. (2003) have
studied the biochemical composition of alginates of algae
harvested from the Egyptian Red Sea coast. They observed that
total carbohydrate and fucose contents were 74.93 and 11.63%,
respectively in C. trinode, 57.87 and 5.62%, respectively in S.
dentifolium; 32.16 and 4.15%, respectively in S. asperifolium
and 42.26 and 8.24%, respectively in S. latifolium.
FT-IR analysis of alginic acid
The FT-IR result indicated that in the region of 3600–1600 cm-
1, five bands appeared with a broad band centered at 3414.74
cm-1. It was assigned to hydrogen bond (O–H) stretching
vibrations, the weak signal at 2929.30 cm-1 due to C–H
stretching vibrations, the wavelength at 2360.18 cm-1 indicated
the presence of R2C=N=N and the asymmetric stretching of
carboxylate O–C–O vibration at 1611.26 cm-1. The band at
1417.64 cm-1 may be due to C–OH deformation vibration with
contribution of O–C–O symmetric stretching vibration of
carboxylate group. The weak bands at 1038.05 cm-1 may be
assigned to C–O stretching, and C–O and C–C stretching
vibrations of pyranose rings. The spectrum showed a band at
887.25 cm-1 assigned to the C1–H deformation vibration of -
mannuronic acid residues. The band at 812.91cm-1 seems to be
characteristic of mannuronic acid residues (R=C=CHR). The
band at 618.68cm-1 may be due to C≡C-H stretching vibration
(Fig. 1). In accordance with these, Leal et al. (2008) have
reported the FT-IR analysis of alginate in three species of
brown seaweeds. They observed a spectral band at 948.5 cm-1,
which was assigned to be the C–O stretching vibration of
uronic acid residues, and one at 888.3 cm-1 assigned to the C1–
H deformation vibration of -mannuronic acid residues and the
band at 820.0 cm-1 seems to be characteristic of mannuronic
acid residues.
Zhang et al. (2008) have reported the -OH groups present in
alginate are clearly seen at 3400 cm-1. They also suggested that
the peaks attributed to the -CH2 groups present at 2931 cm-1
and 2926 cm-1 in alginate and some distinct peaks such as
carboxyl group showed strong absorption bands at 1614 cm-1,
1416 cm-1 and 1306 cm-1, due to carboxyl anions. The band at
1648 cm-1 is attributed to the absorption band of the carbonyl (-
HC=O) stretching. The other band at 1041 cm-1 that was
assigned to the stretching vibration of (CH-OH) appeared at
1643 cm-1 and 1045 cm-1 for the composite gel beads.
13C and 1H NMR analysis of alginic acid
1H NMR and 13C spectroscopy is a reliable method for the
determination of the composition and also the block structures
of alginate molecules (Larsen et al., 2003). The results on 13C
and 1H NMR spectral analysis of purified alginic acid are
given in the Fig. 2 and 3. The 13C NMR spectrum showed
absorptions corresponding to a β-D mannuronic acid at ppm
99.89 (C-1), 66.88 (C-2), 69.79 (C-3), 79.97 (C-4), 75.53 (C-5)
and 175.34 (C-6). Similarly, the 1H NMR spectrum showed the
correlation of these signals with the ppm of 4.248 (H-1), 3.830
(H-2), 3.693 (H-3), 3.987 (H-4), 3.721 (H-5) and 1.005 (H-6),
respectively. Similarly the 13C NMR spectrum showed sharp
absorptions corresponding to a guluronic acid at ppm 16.58,
57.11, 64.62, 131.17 and 165.53 respectively for C1, C2, C3,
C4, C5 and C6. The 1H NMR spectrum showed the correlation
of these signals with the ppm of 0.970, 3.432, 3.450, 3.467 and
3.485 respectively for H1, H2, H3, H4 and H5, respectively.
Similarly, Torres et al. (2007) have reported that the NMR
analysis of S. vulgare alginate and they observed the guluronic
acid anomeric proton (G-1) at 5.06 ppm; guluronic acid H-5
(G-5) at 4.4 ppm; and mannuronic acid anomeric proton (M-1)
and the C-5 of alternating blocks (GM-5) overlapped at 4.7
ppm. Larsen et al. (2003) have studied the NMR spectrum of
alginates from algae harvested at the Egyptian Red Sea coast.
They reported that the presence of gluronic acid and
manuronic acid protons at 3.50 – 3.56 and 3.80 – 4.80ppm,
respectively and the carbon at 71.2 – 72.4 and 93.4 – 94.8ppm
in S. asperifolium alginate.
Figure 1. FT-IR analysis of alginic acid extracted from brown seaweed S. wightii
1867 International Journal of Current Research in Life Sciences, Vol. 07, No. 04, pp.
1863-1872, April, 2018
Figure 2. 13C NMR analysis of alginic acid extracted from brown seaweed S. wightii
Figure 3. 1H NMR analysis of alginic acid extracted from brown seaweed S. wightii
Figure 4. Cumulative mortality percentage of shrimp P. monodon PL fed on different concentrations (100 – 400mg L-1) of alginic
acid enriched Artemia nauplii after bath challenged with WSSV in 21 days interval. Each value is the Mean ± SD of three
replicates
1868 International Journal of Current Research in Life Sciences, Vol. 07, No. 04, pp.
1863-1872, April, 2018
Cumulative mortality percentage during WSSV Challenge
study
After WSSV challenge test, the P. monodon PL were
succumbed to death started from 3rd day, on this day 8 %
mortality was observed in control group. At the same time, no
mortality was observed in experimental groups. The
experimental groups with lowest concentrations (100 and 200
mg L-1) of alginic acid enriched Artemia nauplii fed PL
showed 5 and 4 % mortality during 4th and 5th days of
challenge experiment, respectively. But the highest
concentrations (300 and 400 mg L-1) of alginic acid enriched
Artemia nauplii fed groups displayed 4 and 2% mortality
respectively on 6th day of challenge experiment. When the
duration of the challenge experiment increased, the cumulative
mortality was also increased progressively. At last, 100%
mortality was observed in control group within 8th day of
challenge test. But in the experimental groups, the mortality of
PL was decreased with increasing concentrations of alginic
acid. Accordingly, the mortality observed was 100, 95, 86 and
79% on 21st day of challenge experiment in 100, 200, 300 and
400mg L-1 concentrations of alginic acid enriched Artemia
nauplii fed PL, respectively (Fig. 4).
Cumulative Mortality Index (CMI) and reduction in
mortality
The CMI of control group was 21,737, but it reduced
considerably to 17.15, 36.21, 43.21 and 49.99 %, respectively
in 100, 200, 300 and 400 mg L-1 concentrations of alginic acid
enriched Artemia nauplii fed shrimp (Table 3). The reduction
in mortality of all the tested groups increased with increasing
concentrations (100 400 mg L-1) of alginic acid. The lower
concentration of alginic acid exhibited lower inhibitory activity
against WSSV. But the higher concentration (400 mg L-1) of
alginic acid showed higher inhibitory activity against WSSV.
Similarly, Immanuel et al. (2012b) have observed that the
P.monodon (PL35) fed on Artemia nauplii enriched with
sodium alginate powder (26.5 to 52.4 %) or beads (35.2 to 58.4
%) of S. wightii experienced on reduction in mortality rates
progressively as alginate concentrations were increased from
100 to 400 mg L−1. Sivagnanavelmurugan et al. (2012) have
also reported the fucoidan of S. wightii enriched Artemia
nauplii fed groups showed increased resistance against WSSV
and they observed the reduction in mortality percentage of
experimental groups of shrimp was ranged from 33.71 to 61.65
%, respectively in 100 to 400 mg L-1 fucoidan enriched
Artemia nauplii fed groups, over control group. Immanuel et
al. (2010) have reported the effect of hot water extract of
brown seaweeds S. duplicatum and S. wightii on WSSV
resistance in shrimp, P. monodon PL. They pointed out that the
mortality percentage of P. monodon PL challenged with
WSSV reduced to a maximum of 39.35 to 65.83 % in S.
wightii groups and 16.12 to 47.92 % in S. duplicatum groups.
In both the seaweed extracts, the reduction in mortality of the
tested groups showed increase with the increasing
concentrations of seaweed extracts. The lower concentration
(250 mg L-1) of both the seaweed extracts showed lower (16.12
and 39.35 %) inhibitory activity against WSSV. But in higher
concentration of 750 mg L-1, both the seaweed extracts showed
higher (47.92 and 65.83 %) inhibitory activity against WSSV.
Chang et al. (1999) and Chang et al., (2003) stated that the β-1,
3 glucan has improved the immunity effectively and increased
the resistance to WSSV in PL and juveniles of P. monodon. In
this study, all shrimps in the WSSV challenged, glucon free
(control) group died within 5 days. But the mean survival in
the WSSV challenged glucon fed group was 12.2 % on 6th day.
Chotigeat et al. (2004) reported that the oral administration of
fucoidan from brown algae S. polycystum has reduced the
impact of the WSSV infection in the tiger shrimp P. monodon.
They pointed out that, 4.4, 14 and 44 % of the shrimp survived
respectively in 5-8 g shrimp fed on the crude fucoidan of 100,
200 and 400 mg Kg-1 of body weight / day before and after
challenged with WSSV. The mechanism of inhibition of the
virus is that the negative charges of the sulfate group of the
alginic acid bind with positive charges of amino acid at V3
loop of viral envelope glycoprotein (gp120). The V3 loop is
essential for virus attachment to cell surface heparin sulfate, a
primary binding, before more specific binding occurs to the
CD4 receptor of CD4+ cell. Therefore, the virus could not
invade into the host cells (Witvrouw & De Clerq, 1997).
RT-PCR analysis: WSSV infected P. monodon PL from all
the experimental and control groups were screened by RT-PCR
analysis for the quantification of WSSV DNA (Table 4 and
Fig. 5).
Table 3. Cumulative Mortality Index (CMI) and percentage reduction in mortality of shrimp P. monodon PL fed on
different concentrations of alginic acid enriched Artemia nauplii after bath challenged with WSSV against control
Seaweed product Concentration (mg L
-1
) CMI Reduction in mortality (%)
Control 21737 ± 240.12
a
0 ± 0
Alginic acid 100 18007 ± 204.41
b
17.15 ± 0.111
200 13866 ± 183.71
c
36.21 ± 0.136
300 12343 ± 163.29
d
43.21 ± 0.130
400 10870 ± 142.88
e
49.99 ± 0.150
Each value is the Mean ± SD of three replicates. Within each column, Means with the different superscript letters are
statistically significant (one way ANOVA, P< 0.05 and subsequently post hoc multiple comparison with Tukey’s test).
Table 4. Quantification of WSSV DNA copies by RT-PCR analysis of shrimp P. monodon PL fed on different
concentrations of alginic acid enriched Artemia nauplii after bath challenged with WSSV.
Seaweed product Concentration (mg L
-1
) Ct FAM (Cycles) No. of DNA Copies
Negative control - -
Positive Control 18.69 2.23 x 10
5
Alginic acid 100 20.41 71757
200 21.34 39014
300 22.18 22442
400 34.73 5.73
1869 International Journal of Current Research in Life Sciences, Vol. 07, No. 04, pp.
1863-1872, April, 2018
The WSSV infection in positive control group showed 2.23 x
105 WSSV DNA copies with in 18.69 threshold cycles (Ct
FAM). But in the experimental groups, the copy number of
WSSV DNA was decreased with increasing concentrations of
alginic acid. The experimental group with low concentration
(100 mg L-1) of alginic acid enriched Artemia nauplii fed
shrimp PL displayed 71757 WSSV DNA copies within 20.41
threshold cycles. But in the 200 and 300 mg L-1 concentrations
of alginic acid enriched Artemia nauplii fed groups, 39014 and
22442 WSSV DNA copies were determined with in 21.34 and
22.18 threshold cycles, respectively. Invariably, the shrimp PL
fed on the highest concentration (400 mg L-1) of alginic acid
enriched Artemia nauplii displayed only 5.73 WSSV DNA
copies within 34.73 threshold cycles. Negative controls did not
show any amplification. Strong linear correlation (R2= 0.999)
was obtained between the threshold cycles (Ct) and the amount
of WSSV DNA copies in RT-PCR with reaction efficiency (E=
0.93) and proper slope (M= -3.495) indicating that the assay
had a large dynamic range (Fig. 6).
According to the RT-PCR result, the WSSV DNA copy
numbers in shrimp PL of P. monodon was decreased with
increasing concentrations of alginic acid. Similarly, Immanuel
et al. (2012a) have quantified the WSSV DNA in shrimp P.
monodon fed on different concentrations (0.1 – 0.3 %) of
fucoidan supplemented diets for 45 days and subsequent
WSSV challenge test for 21 days. They observed that the
positive control group had 1.42 x 106 WSSV DNA copies
within 16.96 threshold cycles (Ct FAM), whereas in the
experimental groups, the copy numbers of WSSV DNA was
positively decreased (756 - 11 within 27.23 - 36.26 threshold
cycles). Zhu and Zhang (2011) quantified the WSSV DNA
copies in WSSV challenged shrimp Marsupenaeus japonicus
treated with antiviral VP28-siRNA. They recorded significant
(P< 0.05) reduction of WSSV DNA copies in treatment groups
than that of positive control (no treatment group).
Conclusion
In conclusion, the present findings clearly demonstrated that
the brown seaweed S. wightii is rich source of gluronic acid
and manuronic acid containing polysaccharide-alginic acid.
Further it emphasized that the shrimp P. monodon postlarvae
which received alginic acid of S. wightii showed increased
resistance against WSSV and also it can be used as a
prophylactic agent to improve survival of shrimp against
WSSV infection in aquaculture practice.
Acknowledgements
The authors thank to University Grants Commission, New
Delhi, India, for financial support through Dr. D. S. Kothari
Postdoctoral Fellowship ((BSR)/BL/14-15/0211).
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*******
... Consequently, these compounds can be used as supplements to stabilize and modulate food consistency, such as in baked food, jams, ice cream, jellies, and mayonnaise [6][7][8][9][10][11]. Regarding the algal polysaccharide biotechnological applications that directly affect humans, especially in biomedical and food areas, the purity of the polysaccharide is a determining factor in order to guarantee the compound's safety in order to be used in food processing. This safety record is mainly determined by analyzing its phytochemical properties and protein content (purity rate or polysaccharide quality level) [12,13]. If the polysaccharide purity rate is high, these polysaccharides show interesting rheological properties [14], as well as biodegradability, biocompatibility, and the absence of toxicity [6]. ...
... The phytochemical characteristics results indicate that the sodium alginate extracted from different brown seaweeds do not have terpenoids, tannins, glycosides, phlorotannins, flavonoids, phenols, steroids, and alkaloids. Similarly, Sivagnanavelmurugan et al. [12] and Kavitha et al. [50] have determined the purity of alginic acid, which indicates the absence of tannins, flavonoids, phenols, steroids, and alkaloids; however, only saponins (due to be moiety with carbohydrates) were found, which supports the high purity of the extracted alginates. ...
... Consequently, these compounds can be used as supplements to stabilize and modulate food consistency, such as in baked food, jams, ice cream, jellies, and mayonnaise [6][7][8][9][10][11]. Regarding the algal polysaccharide biotechnological applications that directly affect humans, especially in biomedical and food areas, the purity of the polysaccharide is a determining factor in order to guarantee the compound's safety in order to be used in food processing. This safety record is mainly determined by analyzing its phytochemical properties and protein content (purity rate or polysaccharide quality level) [12,13]. If the polysaccharide purity rate is high, these polysaccharides show interesting rheological properties [14], as well as biodegradability, biocompatibility, and the absence of toxicity [6]. ...
... The phytochemical characteristics results indicate that the sodium alginate extracted from different brown seaweeds do not have terpenoids, tannins, glycosides, phlorotannins, flavonoids, phenols, steroids, and alkaloids. Similarly, Sivagnanavelmurugan et al. [12] and Kavitha et al. [50] have determined the purity of alginic acid, which indicates the absence of tannins, flavonoids, phenols, steroids, and alkaloids; however, only saponins (due to be moiety with carbohydrates) were found, which supports the high purity of the extracted alginates. ...
... Consequently, these compounds can be used as supplements to stabilize and modulate food consistency, such as in baked food, jams, ice cream, jellies, and mayonnaise [6][7][8][9][10][11]. Regarding the algal polysaccharide biotechnological applications that directly affect humans, especially in biomedical and food areas, the purity of the polysaccharide is a determining factor in order to guarantee the compound's safety in order to be used in food processing. This safety record is mainly determined by analyzing its phytochemical properties and protein content (purity rate or polysaccharide quality level) [12,13]. If the polysaccharide purity rate is high, these polysaccharides show interesting rheological properties [14], as well as biodegradability, biocompatibility, and the absence of toxicity [6]. ...
... The phytochemical characteristics results indicate that the sodium alginate extracted from different brown seaweeds do not have terpenoids, tannins, glycosides, phlorotannins, flavonoids, phenols, steroids, and alkaloids. Similarly, Sivagnanavelmurugan et al. [12] and Kavitha et al. [50] have determined the purity of alginic acid, which indicates the absence of tannins, flavonoids, phenols, steroids, and alkaloids; however, only saponins (due to be moiety with carbohydrates) were found, which supports the high purity of the extracted alginates. ...
Article
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Alginates are one of the most important compounds of brown seaweeds. These compounds are employed in the food area, because of their important rheological properties, such as viscosity, gelling, and stabilizing features and as dietary fiber source. In this study, five species of dominant brown seaweeds were collected in the Red Sea (Padina boergesenii, Turbinaria triquetra, Hormophysa cuneiformis, Dictyota ciliolata, and Sargassum aquifolium) so as to characterize the alginate yield and its properties. The analysis demonstrated differences in the alginate yield among the seaweeds. The highest yield of alginate was recorded in the species T. triquetra (22.2 ± 0.56% DW), while the lowest content was observed in H. cuneiformis (13.3 ± 0.52% DW). The viscosity from the alginates varied greatly between the species, whereas the pH varied slightly. The alginate exhibited a moisture content between 6.4 and 13.1%, the ash content ranged between 12.3 and 20% DW, the protein reached values from 0.57 to 1.47% DW, and the lipid concentration varied from 0.3 to 3.5% DW. Thus, the phytochemical analysis demonstrated that the extracted alginates can be safely applied in the food industry. Furthermore, the alginate yield reveals the potential application of these seaweeds as a nutraceutical raw source, which can be exploited by the food industry. Keywords: alginate; dietary fiber; Red Sea; physicochemical properties; phytochemical proper�ties; nutrition
... The alginic acid was extracted using the modified method of Sivagnanavelmurugan, Radhakrishnan, Palavesam, Arul and Immanuel (2018). The milled seaweed were added to a solution of HCl (Fisher Chemicals, Portugal) at 1.23% (1:30 v:v) (3 mL of HCl: 87 mL of distilled water per 3 gs of dried seaweed) was added and kept at room temperature (23°C) for 48 h. ...
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... The extraction of alginic acid was performed in triplicate, employing the adjusted method of Sivagnanavelmurugan et al. [143]. Milled seaweed was added to a solution of HCl at 1.23% (1:30 v:v) and kept at room temperature for 48 h. ...
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