Anticoagulant activity of chondroitin sulfates from Persian sturgeon
(Acipenser pesicus) cartilage
Katayoon Karimzadeh *1, Saeid Delshad 1 and Ali Mostafaie2
1-Department of Marine Biology, Lahijan Branch, Islamic Azad University, Lahijan, Iran
2-Department of Immunology, School of Medicine,Kermanshah University of Medical Sciences,
Kermanshah,Iran
Authors
1-Katayoon Karimzadeh: Corresponding author, supervisor, Department of Marine Biology,
Lahijan
Branch, Islamic Azad University, Lahijan, Iran. [email protected]
2-Saeid Delshad: Ms students, Department of Marine Biology, Lahijan Branch, Islamic Azad University,
Lahijan, Iran. [email protected]
3-Ali Mostafaie: Co supervisor, Department of Immunology, School of Medicine,Kermanshah University of
Medical Sciences, Kermanshah, Iran. [email protected] or [email protected]
Abstract
Chondroitin sulfate (CS) with anticoagulant activities was isolated from Persian
sturgeon
(Acipenser
persicus)
cartilage.
The
enzymatic
extraction,
trichloroacetic acid for deporteinzation, alcohol precipitation and ion exchange
chromatography were conducted to extract
sturgeon CS. Anticoagulant
activities were determined by an activated partial thromboplastin time (APTT),
thrombin time (TT) and prothrombin time (PT) indicators. The extracted
sturgeon CS had the average molecular weight of 45kDa and it was mainly
composed of chondroitin 4 sulfate (C4S) according to the infrared spectra. The
Persian Sturgeon cartilage CS could significantly extend the APTT and TT.
Therefore, Sturgeon CS showed an intrinsic inhibitory mechanism in blood
coagulation pathways. The extracted CS can be used as a potent anticoagulant
agent.
Key words: Anticoagulant, chondroitin sulfate, cartilage, Persian sturgeon
Introduction
Chondroitin sulfate (CS) is an acid mucopolysaccharides, which is mainly
found in the extracellular matrix, especially cartilaginous tissue of mammalian
and some invertebrates and bacteria, as well (Shi et al., 2014; Malavaki et al.,
2008). CS is a polymeric carbohydrate which is composed of repeating
disaccharide units of glucuronic acid (GlcA) and N-acetyl galactosamin
(GalNAc) which links β-(1 → 3) glycoside and sulfate bond in different carbon
position (Khuda et al., 2015; Im et al., 2010).
CS is categorized to the different types in terms of sulfate group position which
its composition is associated with the organisms and type of tissue that is
extracted from. Terrestrial and marine sources of CS have revealed different
chain lengths and several sulfated disaccharides (Shi et al., 2014). For example,
ChSD in shark, ChSA and ChSC in turbot, Chs E in salmon can be mentioned
(Garnjanagoonchorn et al., 2007). CS is a molecule with a wide range of
biological activities, with pharmaceutical, cosmetic and health application
(volpi , 2007).
Regarding the anti-inflammatory, anticoagulant and anti-thrombus activities of
CS, it can be helpful for improving pathological joint discomfort and also it has
an important role to treat diseases caused by dysfunction of the blood
circulatory system. (Gui et al., 2015; Sugahara et al. ,2003). The anticoagulant
activity of CS extracted
from cartilage and bone of some fish species
particularly bony fishes, sharks and Sturgeon have reported (Im, et al.,2010;
maccari et al.,2010 ;Chu, et al., 2015 )
Most available commercial glycos aminoglycan(GAGs)have originated from
animal sources such as Sheeps, pigs, chickens keel and other mammalian tissues
(Vazquez, et al., 2013; ). Due to the incidence of bovine spongiform
encephalopathy (BSE), avian influenza among animal resources, searching
conceivability of CS in marine surceases have been considered (Maccari et
al.,2010). Acipenser perscicus , is one of the most valuable commercial fish
which is residing in the south of Caspian Sea, Iran. Because of increasing
demand for caviar and meat, sturgeon farms were currently developed
(Pourkazemi , 2007; Krylova , 1999 ). So, large amounts of waste produced
during fisheries processing which can be utilizing for pharmaceuticals,
nutraceutical and therapeutic purposes (Volpi, 2009). Therefore, the aim of this
study was to isolate and characterize CS from Persian sturgeon and to evaluate
anticoagulant actives of extracted CS.
Material and Methods
Chondroitin4- sulfate from bovine trachea and chondroitin-6- sulfate from shark
cartilage were obtained from Fluka. Sturgeon cartilage was purchased from
local sources. All other used chemicals and reagent were of analytical grade.
Preparation of Sturgeon cartilage
Sturgeon cartilage was cleaned by tap water and Phosphate-buffered saline
(PBS), respectively. Then, it was chopped, lyophilized and grounded in the
blender to obtain the dried powder.
Enzymatic extraction of CS from sturgeon cartilage
Cartilage powder, approximately 5g was defatted by acetone and afterwards
was digested by adding trypsin at 1:10 (w/v) in PBS buffer containing 0.01%
sodium azide. Then, the mixture was kept for 18h at 65°C until the clear
solution was observed. The trichloroacetic acid (TCA) with 6.1 M was added
to solution and kept overnight at 4°C. For precipitation of protein, centrifuge
was done at 55000 g for 10 min at 4°C. The obtained supernatant was
precipitated with 80 ethanol (v/v). The sample was centrifuged at 4000gfor 30
min at 4°C and the pellet was kept for further experiments (Maccari et al.,
2010).
Extraction by Ion Exchange Chromatography
In order to extraction of CS, the pellet was dissolved in 50mM NaCl and
applied on DEAE –Sepharose Cl-6B anion exchange column which was
equilibrated with 50 mM NaCl. The column was eluted with NaCl gradient
concentration (0.5-1.2 mM) at flow rate of 1 ml/min. Carbazole reaction method
was used to determine the uronic acid content in the fractions which were
eluted. Fractions with high amount of uronic acid contents were pooled and
freeze dried as the CS purified sample.
Carbazole Assay
Carbazole test was used to assess the uronic acid concentration of column
fractions using Heparin as the standard (100, 200, 400 μg/ml). 50 μl of each
sample (1-26 separated fractions) was added to 96 well plates. 200 μl of sodium
tetraborate (25 mM) in sulfuric acid was added. The mixtures were incubated at
100°C for 10 min. Carbazole (50μΜ of 0.125%) was added to each well and
was incubated again at 100°C. Adsorption was read at 550 nm by microplate
reader and the standard curve was plotted (Cesaretti et al., 2003).
PAGE electrophoresis
Electrophoresis was used in 5 % and 7.5% acrylamides in Tris buffer at pH 8.8.
Samples were dissolved in deionized water and separated in Tris-glycine buffer
at pH 8.3 (electrode buffer) for 30 min at a voltage of 50 and 150 V for 90 min.
Alcian blue was used for staining the PAGE electrophoresis.
FT-IR analysis
The structure of Sturgeon CS was carried out through infrared spectrometer at a
wavelength of 400 to 4000 cm-1 (Fourier transform infrared spectroscopy
(FTIR) 8400S spectrophotometer, Japan). The sample was entirely mixed with
KBr at a weight ratio of 1: 100 to prepare a disk.
GAGs assay
To determine the CS amount, we used GAGs assay according to the Fandale
method using 1, 9 Dimethylmehylene blue as substrate and Chondritin-4-sulfate
(C4S) and chondroitin -6- sulfate (C6S) as the standard (Farndale et al., 1982).
The reaction was monitored at 525 nm using spectrophotometer (Jenway, UK).
The amount of C4S and C6S were calculated from calibration curves.
Anticoagulant assay
Human plasmas were collected from healthy individual donors into the tube
with 2.5% sodium citrate solution. The plasma was obtained by centrifugation
at 5400 g for 30 min at 4°C. Afterwards, the plasma was kept at -60°c until use.
Anticoagulant activity of the extracted Cs was assayed by activation with some
factors including partial thromboplastin (APTT), prothrombin time (PT),
Thrombin time (TT). The 10µ extracted CS of different concentrations (1, 2, 4
mg/ml) was mixed with 90 µl of human plasma, and incubated at 37°C for 1
min. Thereafter, 100µl of APTT reagent was added to the samples and
incubated at 37° C for 10 min. Then, the clotting time was measured after
adding 100µl CaCl2 (0.05) by a coagulometer (SY-B032, SUNNYMED) using
heparin and PBS buffer as the standard and negative control, respectively
(Sanderson
et al., 2004).
To determine the PT indicator, plasma was mixed with 10µl of the samples and
incubated at 37°C for 10 min. Then, 200 µl of pre incubated PT reagent was
added and the clotting time was measured. In the TT assay, 190µl human
plasma was mixed with 10µl of the sample and was incubated at 37°C for
2min.Then 100µl of PT assay reagent was added and clotting time was
measured.
Statistical analyses
All data were expressed as mean ± standard deviation (SD) and statistical
analyses were performed with the SPSS 17 software package. Significance of
results was tested by an analysis of variance (ANOVA) and Duncan's MultipleRange Test. Significance of differences was defined at p < 0.05.
Results
The presence of Sturgeon CS was evaluated by carbazole test through the uronic
acid estimation at 550nm using calorimetric assay. Among all the fractions, 119 revealed the more amounts of uronic acid (fig.1).
Further, all fractions with high amounts of uronic acid were pooled together as
extracted Sturgeon CS. It was applied on non-reducing poly acrylamide gel
electrophoresis and stained with Alcian blue. The band with molecular weight
of 40-45 kDa in comparison with heparin (5mg/ml) as standard was revealed
(fig. 2). The mean concentrations of C4S and C6S in the extracted CS from
sturgeon cartilages were calculated as 5.81% and 4.28% using calibration
curveswhich were plotted between the known concentrations (1, 2, 3, 4, 5, and 6
μg/ml) of the standard C4S and C6S at 525nm showed a linear correlation(data
was not shown).
FTIR analysis of CS from sturgeon revealed a pattern similar to the standard
C4S chondroitin sulfate, and proved a strong OH band at 1620–1660/cm(fig.3).
The presence of two medium size bands at 1378 and 1413/cm (O–C=O
bending) showed uronic acids. More absorbance at 239/cm and at 821/cm were
assigned to the stretching of S=O and C–O–S (sulfate group).
The anticoagulant activities of Persian sturgeon CS was measured by three
coagulation indicators,APPT, PT and TT using saline as negative control. The
anticoagulant activities of sturgeon CS in different concentrations were lower
than CS from shark cartilage in terms of APPT and TT (fig. 4 and
6).Concentration dependent was observed in both anticoagulant factors. So that,
the addition of 4 mg/ml of sturgeon CS lead to significant prolongation of the
clotting time (2 and 1.5folds greater than control in terms of APTT and TT
respectively) (p<0.05).The prothrombin time (PT assay) is applied to detect
bleeding disorders. As shown in fig. 5, CS from sturgeon and shark cartilage
were not significant effect on PT indicator (p>0.05).
Discussion
Recently, isolation and purification of CS from marine organisms and
particularly fish species have been greatly considered, duo to the position of
sulfation group and safety of those resources rather than mammalians (Shi et al.,
2014).
In order to extract CS from Persian Sturgeon, enzymatic digestion using trypsin,
protein precipitation by TCA and ion exchange chromatography were
conducted. The obtained Persian Sturgeon CS was estimated about 6.8%.
However, the obtained CS isolated from skull and backbone of Hybrid Sturgeon
(Acipenser baeri X acipenser schrenckii) was recorded as19.5% and 22 % when
Alcalase was used for digestion. (Chu et al., 2015). In another similar study on
CS extraction from Sturgeon (Acipenser sinensis), the obtained cartilage and
backbone CS were measured as 10.5% and 13%, respectively (Im et al., 2010).
The decreased yield of Persian Sturgeon CS is due to the using trypsin in
digestion of cartilage. Unlike Alcalase and papain (exogenous enzymes),
endogenous enzyme like trypsin can produce smaller peptide fragments rather
than exogenous ones in digestion. Therefore, CS can be attached to those
fragments and be removed during TCA precipitation (Yang et al., 2010 ).
Although the autolysis of GAGs by endogenous enzyme in the tissue is
possible, but it seems that using exogenous enzymes can be more effective
(Nakano, 2010 ).
The amount of C4 and C6S extracted from Persian Sturgeon using GAGs assay
were estimated 5.81% and 4.28%, respectively. In the Shark fin cartilage, CSs
were quantified as 9.60% and 8.76%, respectively with this method (Xia et
al.,2014 ). However, disaccharide assay using chondroitinase ABC and SAXHPLC chromatography can alternatively be applied for quantification of the
CSs extract. The amount of C4S and C6S in sturgeon (Acipenser sinensis) and
hybrid Sturgeon (Acipenser baeri X acipenser schrenckii) were recorded 88.8%
, 7.2% ,and 37.8%, 59.6% respectively (Chu et al., 2015; Im et al., 2010).
Therefore, the amount of C4S was more than C6S in Persian Sturgeon and its
disaccharides pattern was similar to CS from Sturgeon (Acipenser sinensis) and
CS shark fin cartilage.
FTIR analysis of standard chondroitin 4-sulfate and chondroitin 6-sulfate
showed a peak at wavelengths of 857 and 826 cm-1. The peak at 857 cm-1 can be
assigned to C4S, while the absorbance at 826 cm-1 can be used to identify C6S
(Garnjanagoonchorn et al., 2007).
The FTIR spectrum of Sturgeon CS showed a peak at a wavelength of 856.39
cm-1, which indicated C4S. This peak (856.39 cm-1) indicated C-O-S bond
(Honda et al., 1974; Foot and Mulholland., 2005 and Garnjanagoonchorn et al.,
2007).
In order to determine the amount of inhibition of the coagulation cascade,
anticoagulant indicators (APTT, PT, TT) were used.The observed promoted
times of APTT and TT with the increasing levels of CS can be showed a proper
intrinsic inhibitory mechanism and thrombin activity with fibrin polymerization
in coagulation pathway by sturgeon CS, respectively. However, Sturgeon CS in
different concentrations does not have a significant effect in PT indicator, and it
can suggest that CS cannot modulate extrinsic inhibitory mechanism.
Anticoagulant activities of the extracted CS in hybrid Sturgeon showed a dosedependent manner in CS concentration, particularly in backbone CS (Chu et al.,
2015). Therefore, anticoagulant activities of CS can be related to the type and
amount of mono-sulfated disaccharide (Majdoub et al., 2009; Fonseca, et al.,
2008). The increased levels of mono-sulfated disaccharide caused a stronger
anticoagulant activity, as it has been recorded in hybrid Sturgeon skull CS
(Mulloy et al., 2000). The blood clotting is a complex mechanism. Accordingly,
further studies can be necessary to evaluate the anticoagulant activity of CS.
Conclusion
It can be concluded that the sturgeon CS can be extracted by enzymatic
digestion (trypsin) and ion exchange chromatography. This extracted CS has a
comparable structure with CS that is isolated by different methods. Sturgeon CS
showed an anticoagulant activity and it is comparable with the commercial
anticoagulants.
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Concentration of Uronic acid (µg /ml)
300
250
200
150
100
50
0
1
3
5
7
9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49
Fraction (2ml)
Fig. 1: Anion-exchange chromatography of the crude polysaccharide fraction of sturgeon cartilage on
DEAE –Sepharose Cl-6B. The column was eluted by sodium chloride 0.5 to 1.2 mol/l, at a flow rate
of 20 ml/h. Fraction (2 ml) was collected and the total carbohydrate assayed using the carbazole
reaction.
Lane 1
Lane 2
Fig. 2: Polyacrylamide gel electrophoresis (PAGE) analysis of chondroitin sulfate extract from
Persian sturgeon cartilage, stained Alcian Blue. Lane 1: heparin as marker, Lane2: CS extracted from
sturgeon cartilage
Fig. 3: FTIR spectrum of chondroitin sulfate extract from Persian sturgeon Cartilage.
70
B
60
c
b
50
APPT (Sec)
B
40
a
A
Control
30
Sturgeon cartilage
20
Shark Cartilage
10
0
1
2
4
Concentration (mg/ml)
Fig. 4: Anticoagulant activity as indicator of APTT of chondroitin sulfates from Persian sturgeon.
Each value is the mean ± SD of triplicate measurements.Different letters indicate significant
differences (p < 0.05)
10.2
A
a
PT (Sec)
10
9.8
9.6
a
a
A
Control
A
Sturgeon
cartilage
9.4
9.2
Shark cartilage
9
1
2
Concentration (mg/ml)
4
Fig.5: Anticoagulant activity as indicator of PT of chondroitin sulfates from Persian sturgeon. Each
value is the mean ± SD of triplicate measurements. Different letters indicate significant differences (p
< 0.05)
35
C
30
B
TT( Sec)
25
20
a
A
c
b
Control
15
Sturgeon cartilage
10
Shark Cartilage
5
0
1
2
4
Concentration (mg/ml)
Fig. 6: Anticoagulant activity as indicator of TT of chondroitin sulfates from Persian sturgeon. Each
value is the mean ± SD of triplicate measurements. Different letters indicate significant differences (p
< 0.05)