Structural Characterization and Biological Activity of Acidic Bacillus polymyxa

Journal of Applied Sciences Research, 3(10): 1170-1177, 2007
© 2007, INSInet Publication
Structural Characterization and Biological Activity of Acidic
Polysaccharide Fractions Isolated from Bacillus polymyxa NRC-A
Mohsen M. S. Asker, Manal, G. Mahmoud and Ghada, S. Ibrahim
Microbial Biotechnology Department, National Research Center, Dokki, Cairo, Egypt.
Abstract: The heteropolysaccharides coded as AP-I, AP-II and AP-III were obtained from the crude
extracellular polysaccharide of Bacillus polymyxa NRC-A by eluting from DEAE-cellulose column and
Sephadex G-150 column. Their chemical structure and molecular weights were characterized by IR, GCMS, periodate oxidation-Smith's degradation and gel permeation chromatography (GPC). These
polysaccharides comprised mainly glucose, mannose, rhamnose and glucouronic acid in the ratio of (3.00:
2.10: 1.40: 1.00), (3.40: 2.52:1.00: 2.11) and (4.11: 2.68: 1.00: 3.00) in AP-I, AP-II and AP-III,
respectively. The antioxidant activities of the AP-I, AP-II and AP-III were evaluated by DPPH radical
scavenging. The order effectiveness of polysaccharide fractions in inhibiting free radicals was as follows:
AP-III>AP-II> AP-I. The effects of the molecular weight and uronic acid content of the polysaccharide
fractions on the improvement of the bioactivities appeared to be significant. In addition, these native and
modified fractions still determined as in-vitro anticoagulant activity. The modified fractions showed high
anticoagulant activity than the native fractions.
Keyword: Exocellular polysaccharide (EPS), Bacillus polymyxa NRC-A, GC-MS, periodate oxidation,
Smith-degradation, antioxidant, anticoagulant.
INTRODUCTION
Microbial exopolysaccharides are produced by
various genera of bacteria and yeast [1 ,2 ] . Many of these
products have been shown to have a wide variety of
applications
in
food,
pharmaceutical and oil
industries [3 ] . Some microorganisms belonging to
Bacillus species have been known to produce
extracellular polysaccharides, such as cellulose [4 ],
levan [5 ], mannan [6 ] and acidic polysaccharides [7 ]. But
Mitsuda's [8 ] produced the EPS composed of glucose,
mannose and glucoronic acid by Bacillus polymyxa No
458. The viscosity of the EPS isolation was 5-8 times
higher than that of locust bean gum. A previous
stud y [ 9 ] s h o w e d th a t an extracellular acid ic
p olysaccharid e p ro duced b y a n ew iso la te d
microorganism, designated as Bacillus polymyxa,
reduced the cholesterol level in serum and liver of
hypercholesterolemic rats [1 0 ]. Recently, some plant
polysaccharides such as pectin, guar gum, and sodium
a l g i n a t e h a v e a ttra c te d a tten tio n fo r th e ir
hypochololesterolemic effect on experimental rats. In
the course of study, it has been found that some strains
of Bacillus sp. were capable of producing new types of
acidic heteropolysaccharides. Reactive oxygen species
(ROS), capable of causing damage to DNA, have been
associated with carcinogenesis, coronary heart disease,
and many other health problems related to advancing
age [1 1 ,1 2 ]. The polysaccharides have been demonstrated
to play an important role in dietary free-radical
scavenging for oxidative damage prevention. There is
an increasing evidence indicating that the reactive
oxygen species produced by sunlight, ultraviolet light,
ionizing radiation, chemical reaction and metabolic
process have a wide variety of pathological effects.
These effects might be DNA damage, carcinogenesis,
and cellular degeneration related to aging [1 3 ,1 4 ]. Thus, it
is essential to develop and utilize effective and natural
antioxidants so that they can protect the human body
from free radicals and retard the progress of many
chronic diseases [1 5 ]. Recently, many natural resources
have attracted attention in the search for bioactive
compounds to develop new drugs and healthy foods.
Some alga polysaccharides have been demonstrated to
play an important role as free-radical scavengers in
vitro and as antioxidants for the prevention of oxidative
damage in living organisms [1 6 ]. Their activity depends
on several structure parameters such as molecular
weights, type of sugars and glycosidic branching
[1 7 ,1 8 ]
. This work was conducted to study the possible
isolation, purification, fractionation and structure
features of the acidic polysaccharide produced by
isolated Bacillus polymyxa NRC-A, in addition to, the
biological activities of these fractions.
Corresponding Author: Mohsen M.S. Asker, Microbial Biotechnology Department, National Research Center, Dokki,
Cairo, Egypt.
Tel.:+2023335982; fax: +2023370931. E-mail address: [email protected]
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J. Appl. Sci. Res., 3(10): 1170-1177, 2007
M ATERIALS AND M ETHODS
I s o la t io n a n d I d e n t i f i c a t i o n o f B a c t e r ia :
Polysaccharide-producing bacteria were isolated from
soil samples collected from different zones. Isolates
were obtained by serial dilution plating on a seed
media containing (g/l), glucose 20.0; CaCO 3 1.0;
NH 4 NO 3 0.8; K 2 HPO 4 0.6; KH 2 PO 4 0.05; MgSO 4 .7H 2 O
0.05; MnSO 4 4H 2 O, 0.1 and Yeast extract 0.1, at
30ºC [1 9 ]. A total of about 100 colonies were isolated,
and the polysaccharide-producing bacteria were
screened for their ability to produce polysaccharide this
was based on colony morphology (mucous and ropy).
A mucous colony was isolated and identified as
Bacillus polymyxa NRC-A. Metabolic characterization
was carried out in Biolog GP2 Micro Plates TM
according to the instructions of the manufacturer
(Biolog, Hayward CA, USA) and evaluated with
Microlog3 Software.
Isolation and Purification of Polysaccharide: Bacillus
polymyxa NRC-A was grown in a liquid seed media
but using the following concentrations (60.0 g glucose;
4.0 g CaCO 3 and 2.0 g NH 4 NO 3 ) by the method of[2 0 ]
under shaking condition; 150 rpm for 3 days. The
culture broth was diluted with water and centrifuged at
5000 rpm for 20 min (Sigma-Laborzentrifugen, 2K 15)
to remove bacterial cells. Trichloroacetic acid (5%) was
added and left overnight at 4ºC and centrifuged at 5000
rpm again. The pH of the clear solution was adjusted
to 7.0 with NaOH solution and dialyzed three times
(3 x 1L). The supernatant was completed to fourvolumes with ethanol 95% and left overnight at 4ºC.
The precipitate was separated by centrifugation at 5000
rpm, washed twice with acetone and dehydrated by
ether. The crude polysaccharide was dissolved in 0.03
M Na 2 SO 4 and precipitated by cetylpyridinium chloride.
The precipitated CPC-Complex was collected by
centrifugation, washed several times with ethanol,
and
dissolved
in 10% NaCl. The solution was
filtered, dialyzed, and concentrated to a small volume.
The purified polysaccharide was precipitated by
ethanol, yielding a white powder of an acidic
polysaccharide [7 ].
Anion-exchange Chromatographic M ethod: The
acidic polysaccharide (100 mg in 5 ml water) was
subjected to anion exchange chromatography on a
column (2.0 x 40cm) of DEAE-cellulose, preequilibrated using phosphate buffer 0.1 M and eluted
with continuous gradient from 0.0 to 4.0 M NaCl in
the same buffer [2 1 ]. Fractions (5 ml) were collected and
an aliquot (0.1 ml) was tested for total carbohydrate by
the phenol-H 2 SO 4 method using Spectrophotometer UV-
2401PC Shimadzu [2 2 ]. The respective polysaccharide
fractions were pooled and dialyzed overnight against
deionizired water and finally lyophilized.
Gel-filtration Chromatography: Each fraction was
subjected to gel permeation chromatography on a
column (2.6 x 50 cm) of Sephadex G-150, which was
pre-calibrated with dextrans (Fluka), of known
molecular weight. The void volume was determined
using dextran blue. The column was equilibrated and
was eluted with 0.1 M NaCl. An aqueous solution of
sample (50 mg) was dissolved in 2.0ml of 0.1 M NaCl
and loaded onto the column bed. Fractions (5.0 ml)
were collected and tested for total carbohydrate by the
phenol-H 2 SO 4 method [2 2 ].
M olecular W eight Determination: The average
molecular weights of AP-I, AP-II and AP-III were
determined by a gel-chromatographic technique.
Standard dextrans 2000000, 700000, and 40000 (Fluka)
were passed through a (2.6 x 50 cm) SephadexG-150
column, and then the elution volumes were plotted
against the logarithm of their respective molecular
weights. The elution volumes of AP-I, AP-II and APIII were plotted in the same graph, and the molecular
weights were determined [2 3 ].
D e te rm in a tio n o f P o ly sacch a rid es S u g a r
Composition: The polysaccharides AP-I, AP-II and
AP-III were hydrolyzed with 2.0 M triflouroacetic acid
(TFA) and the tubes were heat-sealed. Hydrolysis was
carried out at 105ºC for 4 h in an oven. After the
hydrolysis, the acid was removed by evaporation on a
water bath at a temperature of 40ºC and codistilled with water (3 x 5 ml) [2 4 ]. The purified
hydrolyzates (20µl) were analyzed by HPLC according
to El-Sayed's [2 5 ].
Infra Red Spectroscopy: The polysaccharides were
also characterized using a Fourier transform infrared in
Bucker Scientific 500-IR spectrophotometer. The dried
polysaccharides were grounded with KBr powder and
pressed into pellets for FT-IR spectra measurement in
the frequency range of 400-4000 cm -1 [2 6 ].
Periodate Oxidation- Smith Degradation: Samples of
AP-I, AP-II and AP-III (16 mg) were added separately
to 50 ml NaIO 4 0.1 M solution in round bottom flasks,
and the mixtures were kept at 4ºC in the dark [2 7 ].
Aliquots (2 ml) were removed after different intervals
of time and consumption of periodate and HCOOH
products were determined [2 8 ,2 9 ]. Ethylene glycol (2 ml)
was added, and then the experiment of periodate
oxidation was over. The solutions were dialyzed against
tap water and distilled water for 48 h, respectively.
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J. Appl. Sci. Res., 3(10): 1170-1177, 2007
The polyaldehyde was reduced with excess of NaBH 4
at room temperature overnight. The reaction was
terminated by addition of ice cold acetic acid (4 N).
The solutions were again dialyzed as described above,
and lyophilized [3 0 ]. The resulting polyalcohol was
hydrohyzed with HCOOH 90% for 5 h. The sugars and
sugar alcohols were analyzed by HPLC according
to El-Sayed's [2 5 ].
M ethylation Analysis: The native polysaccharide
fractions (AP-I, AP-II and AP-III) and their carboxylreduced by the method of Taylor [3 1 ] (AP-IR, AP-IIR
and AP-IIIR) were methylated separately using the
method delete [3 2 ]. The methylated products were
extracted by CHCl 3 . The product was then hydrolyzed
with 90% HCOOH (5ml) for 5h, and excess HCOOH
was evaporated by co-distillation with distilled water.
The hydrolyzed product was reduced with NaBH 4 (24
mg), and acetylated with pyridine and acetic acid [3 3 ].
The alditol acetates of the methylated sugars were
analyzed by GC-M S Finnegan SSQ-7000 instrument
using a fused silica capillary column (DB-5, 0.25 mm
ID, 30 m). A temperature programming of 60-280ºC
increased by 4ºC/min was maintained for the analysis.
Ionization potential was 70ev and mass range
(m/z) was 40-4000amu. Helium was the carrier gas
used. Qualitative and quantitative identification of the
methylated sugars were determined by comparing
retention time and mass fragmentation patterns with
those of the available authentic data base.
M easurement of Anti-oxidation Activity: For the 2,2 / diphynel-1-picrylhydrazyl (DPPH) assay, 10 mg (in 100
µl saline) each sample was added to 900 µl of DPPH
solution (Freshly prepared at a concentration of 10 -4 M )
and the mixtures were mixed for 10 sec at ambient
temperature 22ºC. The decrease in absorption at 515
nm was measured against a blank of ethanol without
DPPH in 1-Cm quartz cells after 10, 20, 30, and 60
min of mixing using Spectrophotometer UV-VIS
2401PC Shimadzu [3 4 ]. Antiradical action toward DPPH
radical was estimated from the difference in absorbance
with or without sample (control) and the percent of
inhibition was calculated from the following equation:
Inhibition %= (Abs. of control – Abs. of test sample)
x 100/ Abs. of control
The samples of each polysaccharide fractions were
analyzed and the main values as well as the (S.E)
were given.
Anti-coagulant Activity: Venous blood was collected
directly into a vessel containing one tenth volume 3.2%
trisodium citrate. The mixture was immediately
agitated, by gentle inversion, centrifuged at 2000 rpm
for 10min. The canary yellow plasma was pooled [3 5 ].
Anti-coagulant activities of the various original and
modified polysaccharide fractions were determined [3 6 ]
and compared with reference sodium heparin.
RESULTS AND DISCUSSIONS
Isolation, Purification and
Composition
of
EPS: After Bacillus polymyxa NRC-A was grown for
three days, the bacterial cells were separated by
centrifugation, and the crude polysaccharide was
precipitated by ethanol from the supernatant. The crude
polysaccharide was dissolved in sodium sulfate, which
w a s fur the r fra c tio na ted b y tre a tm e nt with
cetylpyridinium chloride (CPC), to give an acidic
polysaccharide fraction (11.3gl -1 ) and a neutral
polysaccharide (3.63gl-1 ) which was kept for further
study. The acidic polysaccharide was purified by
repeated fractional precipitations with ethanol, and upon
chromatography on DEAE-Cellulose (Pharmacia) gave
three fractions: AP-I, AP-II and AP-III (eluted with
0.01 M phosphate buffer containing 0.0 to 4.0 M
NaCl) (Fig. 1). The three fractions AP-I, AP-II and
AP-III were purified by gel filtration chromatography
on Sephadex G-150 column (2.6 x 50 cm) showing a
single and symmetrical peak, indicating that the each
fraction had been purified to homogeneity (Fig. 2-4).
The molecular weights of the polysaccharide fractions
AP-I, AP-II and AP-III were determined by a gel
filtration
technique
using
different
Dextran
markers passing through a Sephadex G150 column
(2.6 x 50 cm), and they were found to be 130 kDa,
110 kDa and 80 kDa, respectively. Analysis of their
acid hyrolyzates by HPLC indicated the presence of
glucose, mannose, rhamnose and glucouronic acid in
the ratios of (3.00: 2.10: 1.40: 1.00), (3.40: 2.52:1.00:
2.11) and (4.11: 2.68: 1.00: 3.00) in AP-I, AP-II and
AP-III, respectively. This indicates that these acidic
Fig. 1: A typical elution profile of the acidic
polysaccharides on DEAE-cellulose column
(2.0x50 cm i.d.) previously equilibrated with
sodium phosphate buffers. A flow rate 0.6 ml/
min and 5.0ml fraction were maintained.
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J. Appl. Sci. Res., 3(10): 1170-1177, 2007
Table 1: M olecular weights and m onosugar m olar ratios of fractions polysaccharide obtained from chrom atographic colum n.
M olar ratios
MWt
--------------------------------------------------------------------------------------------------------------------------Fraction
KD a
Glucose
M annose
Rham nose
Glucouronic acid
AP-I
130
2.10
1.40
1.00
3.00
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------AP-II
110
3.40
2.52
1.00
2.11
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------AP-III
80
4.11
2.68
1.00
3.00
Table 2: M olar ratios of sugar alcohols and im m une sugar in the Sm ith 's degradation hydrolysate.
Sugar alcohols
Fraction
---------------------------------------------------------------------------------------------------------------------------
AP-I
Sugar im m une
----------------------
Erythretol
Erythric acid
Glycerol
Glyceric acid
Rham nose
4.0
0.2
1.0
0.0
0.7
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------AP-II
3.8
0.2
1.3
0.0
1.0
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------AP-III
4.3
0.3
2.6
0.0
1.0
Table 3: Linkage analysis of the constituent sugars of the fractions AP-I, AP-II and AP-III and their carboxyl- reduced fraction AP-IR, AP-IIR
and AP-IIIR.
M olar ratios
M ethylated
M ode of
-----------------------------------------------------------------------------------------------------------------------alditol acetates
linkage
AP-I
AP-IR
AP-II
AP-IIR
AP-III
AP-IIIR
2,3,6-M e 3 -Glc
--4) Glc (13.4
6.1
3.2
7.3
4.6
8.7
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2,3-M e 2 -Glc
-4,6) Glc (12.0
1.9
1.8
1.9
1.6
1.7
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2,3,4,6-M e 4 -Glc
Glc (1--1.0
1.2
1.0
1.0
1.0
1.0
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2,4-M e 2 -Rha
--3) Rha (11.0
1.0
1.0
1.0
1.0
1.0
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2,3,6-M e 3 -M an
-4) M an (11.6
1.7
1.9
2.1
1.9
1.8
Fig. 2: Gel filtration for the chromatography of AP-I
on
Sephadex G-150 (2.6x50cm i.d.), the
column was equilibrated with 0.1 M NaCl at
flow rate 0.5 ml/min and 5ml fraction.
Fig. 3: Gel filtration for the chromatography of AP-II
on Sephadex G-150 (2.6x50cm i.d.), the
column was equilibrated with 0.1 M NaCl at
flow rate 0.5 ml/min and 5 ml fraction.
polysaccharide fractions have the same chemical
composition in spite of the difference in the
glucouronic acid ratio (see table 1). The FT-IR Spectra
of AP-I, AP-II and AP-III showed adsorption bands at
704.9, 788.5, 845.7, 891.7, 1069.7, 2921.7 and 3423.3
Cm -1 in the IR spectrum (Fig. 5). The band at 891.7
Cm -1 was ascribed to be â-type glycosidic linkages in
the polysaccharide [6]. The bands at 891.7 and 932.1Cm -1
were characteristic of (1--4)-â-glycosidic linkages. The
broad band at 16391Cm -1 was due to bound water [3 7 ].
The IR spectra indicated the presence of â-type
glycosidic linkages in the AP-I, AP-II and AP-III.
Periodate Oxidation and Smith Degradation: The
AP-I, AP-II and AP-III were submitted to periodate
oxidation, borhydride reduction and hydrolysis by
heating with 90% HCOOH for 5 h (Smith degradation).
The AP-I, AP-II and AP-III consumed 0.76, 0.73 and
0.84 mol of oxidant per mol of glycosyl residue,
respectively and a small amount of formic acid was
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J. Appl. Sci. Res., 3(10): 1170-1177, 2007
Fig. 4: Gel filtration for the chromatography of AP-III
on Sephadex G-150 (2.6x50cm i.d.), the
column was equilibrated with 0.1 M NaCl at
flow rate 0.5 ml/min and 5 ml fraction.
liberated. The HPLC analysis of the Smith's degraded
polysaccharides, the erythretol, erytheric acid, and
glycerol in addition to rhamnose was determined.
The molar ratios of these sugar derivatives are
shown in (Table 2).
Erythritol was produced from (1--4)-glycosidic
linkages of glucose and/or mannose. W hile, erythric
acid was liberated from (1--4)-glycosidic linkages of
glucouronic acid. Glycerol detected in the polyalcohol
hydrolyzate
indicated
the presence of (1--4)
glycosidic linkages glucose and/or mannose at the
non-reducing end. The disappearance of glyceric acid
through the hydrolysis of the polyalcohol means
that glucouronic acid did not exist at the non-reducing
end, but probably was found inside the backbone [3 8 ,3 9 ].
The appearance of rhamnose as the immune unit
indicated the presence of some (1--3) glycosidic
linkages
rhamnose [4 0 ,4 1 ].
Thus,
the
periodate
oxidation and Smith's degradation study confirmed
the
mode
of linkages,
which
agrees
with
presence of the IR bands [4 2 ].
Linkage Analysis: To obtain some definite information
on the mode of glycosidic linkages of these sugar
residues, the polysaccharide fractions were methylated
and then the methylated sugars were converted to the
corresponding acetylated derivatives. As shown in
(Table 3) glucose, mannose and glucouronic acid
residues in the main chain of the polysaccharide are
linked mainly through (1--4) and (1--6) glycosidic
linkages, as indicated by the presence of 2,3,6 triO-methyl glucose, 2,3,6 tri-O-methyl mannose, and
2.3 di-O-methyl glucose.
Scavenging effect of polysaccharide fractions during D PPH test as m easured by changes in absorbance at 515 nm .
Scavenging (% ) *
--------------------------------------------------------------------------------------------------------------------------------------------------------Fractions
10 m in
20 m in
30 m in
60 m in
AP-I
19.166±0.785
21.796±1.075
29.689±0.243
32.906±0.496
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------AP-II
19.583±0.420
23.742±0.730
39.246±0.741
43.276±0.190
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------AP-III
18.229±0.445
27.933±0.245
59.302±0.379
63.011±0.478
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------D extran
14.193±0.358
17.460±0.253
21.542±0.528
21.970±0.485
* Values are m eans ± S.E of three determ inations.
Table 4:
Fig. 5: Infrared spectra of acidic polysaccharide fractions AP-I, AP-II, and AP-III isolated from B. polymyxa
NRC-A
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J. Appl. Sci. Res., 3(10): 1170-1177, 2007
In addition, the presence of 2,4 di-O-methyl
rhamnose suggests that rhamnose was present in the
main chain and linked mainly by (1--3) glycosidic
linkages.
The presence of 2,3,4,6 tri-O-methyl glucose
indicated that the glucose may be found at the
non-reducing end. As shown the polysaccharide has a
highly branched structure and the side chains are
terminated with glucose. In addition, the presence of
2,3 di-O-methyl glucose suggests that some of the
(1--4) glycosidic linkage glucose residues might come
from a branch point. As regard the glycosidic linkages
of glucouronic acid residues, the methylated carboxylreduced polysaccharides factions gave about two-fold
molar ratios of 2,3,6-tri-O-methyl glucose than native
polysaccharide factions, indicating that glucouronic acid
residues are linked manly by (1--4) glycosidic linkage.
These agree with appearance of glycerol in smith
degradation results. These results agreed with the
appearance of rhamnose as immune units in the Smith's
degradation results [7 ,2 4 ].
Radical Scavenging Ability: Using the same perweight b asis the antirad ical p erfo rma nc e of
polysaccharide fractions with respect to DPPH radicals
was m easured and compared. T he order of
effectiveness of polysaccharide fractions in inhibiting
free radicals was as follows: AP-III>AP-II> AP-I.
Table 4 shows that during the test periods, AP-III had
the highest radical scavenging activity, followed by
AP-II. After 60 min incubation, 63.01% of DPPH
radical were quenched by fraction AP-III, followed by
fraction AP-II which was able to quench 45.3%.
Surprisingly, inhibition of DPPH radicals was only
21.9% pure dextran (Sigma) was assayed.
The results suggested that the molecular weights of
polysaccharide fractions played an important role on
their bioactivity. In the study of acidic polysaccharide,
we obtained three fractions, and found they exhibited
antioxidant ability depending on the uronic acid
content. W ith increased of the uronic acid content,
antioxidant activity of the three fractions increased. The
existence of uronic acid might affect the physicochemical properties of the polysaccharides and hence
their bioactivities. In this study, found that both uronic
acid content and molecular weight of acidic
polysaccharide fractions could play on important role
in the antioxidant activity. Among the acidic
polysaccharides, a relatively low molecular weight and
a high uronic acid content appeared to increase
the antioxidant activity. The results were similar to
Chen's reports that molecular weight was very
important to the antioxidant activity of Green Tea
Table 5: V isual evolution of the
fractions polysaccharide
Fractions
N ative
AP-I
(2+)
AP-II
(2+)
AP-III
(4+)
N a-H eparin
(5+)
*polysaccharide=2.0m g per tube
(5+)= Plasm a clotting after 25m in.
(3+)= Plasm a clotting after 15m in.
(1+)= Plasm a clotting after 5m in.
in-vitro anticoagulant activities of
com pared with sodium heparin.
Polyaldehyde
Polyalcohol
(2+)
(3+)
(3+)
(4+)
(3+)
(5+)
(4+)= Plasm a clotting after 20m in.
(2+)= Plasm a clotting after 10m in.
polysaccharide fractions [4 3 ]. Polysaccharide extracts
from mushroom [1 4 ] and Keisslerilla sp. Y54108
exopolysaccharide [4 4 ] were also reported to have
free-radical scavenging effects related to it’s affinity to
the radical in the specific site. However, the
m e cha nism o f fr e e -r a d ic al sc ave nging o f
polysaccharides is still not fully understood [1 4 ]. It is
known that the phenolic compounds from the plant
Pedicularis alashanica, such as phenylproponiod
glycosides, may react with superoxide radical by a oneelectron transfer mechanism or hydrogen abstraction
mechanism to form the semiquinones. Therefore,
the scavenging
activity
of
their
reduction
may
be related
to
the number of phenolic
hydroxyl
groups
and
the
conjugated
system [4 5 ]. However, it was not clear whether the
mechanisms of radical scavenging by polysaccharide
fractions were similar to that of plant phenolic
compounds or not.
Anti-coagulant Activity: Plasma was coagulated in the
blank-tube after 5 min and in the sodium heparin
containing tube after 25 min. In experimental tubes, the
highest anticoagulant activity was exhibited by AP-III
native and AP-III polyalcohol as shown in (Table 5).
The APIII containing negatively charged COOglucouronic acid, also opening the pyranose rings by
periodate treatment (Polyaldehyde and polyalcohol).
Having the ability of binding Ca + 2 , therefore they
prevent clotting formation [4 1 ,4 6 ,4 7 ].These promising results
can be regarded on initiatory steps towards the
utilization of acidic polysaccharide and modified as
future cheap sources for production of valuable drugs
for treatments of blood coagulant.
ACKNOW LEDGEM ENTS
W e are most grateful to Dr. Y. M. Ahmed Heat of
microbial Biotechnology Department, National Research
Center Cairo Egypt and various members of our
laboratories for the kind supply of different fine
chemicals and giving us the opportunity to complete
this research.
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J. Appl. Sci. Res., 3(10): 1170-1177, 2007
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