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Physiological Activity of New Heparinoids Derived from Plant Polysaccharides.

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New Heparinoids from Plant Polysaccharides
363
Physiological Activity of New Heparinoids Derived from Plant
Polysaccharides
Volker Bode and Gerhard Franz’”
Departmentof Pharmacy, University of Regensburg. D-8400Regensburg, FRG
Received April 24,1990
DerivatiSatiar and anticoagulant effects of thnx heparinoid model substances anz described which are derived from thw different types of plentpolysaccharides. Degree of sulphatationand stluctulal prerequisitesfor inhibiting
coagulationare discussed
Phydologische Akltvlten Reupptiepr Heparinoide aus pclanzlichen Po.
lyepechprklen
Polysaccharides are increasingly used for medical applications, either
taking advantage of some intrinsic functional properties, or of some pharmacological pmperties elicited by biological interactions. Biological and
phannacological properties can be induced or modified in such a polymer
by chemical denvatisation.
Thus,for example the therapeutic efficacy of heparin and heparinoids is
extensively studied by a control of the molecular size,the degree of sulphatation and the localiition of specific sugar or WNC acid sequences.
After a sulphatation of the glucan with chlorosulphonic
acid in pyridine a polymer was obtained with a sulphur
content of 12.5% corresponding to a DS of 0.92. The average M W determined by GPC was shown to be 42 OOO D. In
order to determine the localisation of the SO3 groups, the
sulphated polymer was methylated, hydrolyzed and acety1 a ~ t ~ 9BY
~ this
) . procedure the ~ ~ p g r o uwere
p s not cleaved
prior to acid hydrolysis. GC-MS analysis of the methylated
monomers obtained after hydrolysis revealed the presence
of terminal glucose and 1,4,6-linked glucose residues,
which are the indication for a sulphatation in position of
C-6 of the glucose backbone.
In this respect studies were undertaken with different
structural types of polysaccharides serving as the base for
the subsequent synthesis of heparinoids.
The commonly utilized heparinoids are highly sulphated
polysaccharides with a sulphur content of more than 15%
and an average MW of approximately 10 OOO D. Both criteria, i.e. DS and DP are responsible for antithrombotic and
possible side effects’).
Starting from several well established or newly analyzed
polysaccharide structures, the derivatives were modified in
both, MW and DS in view of their possible antithrombotic
activities.
Durch chemisehe Derivatisierungdreier untaschiedticher pflanzlicher Polysaccharide wurden Heparitwide gewonnen, die auf ihre spezilischen Eigenschaften als Antikoagulans untersucht wurden. Die physiologischenWirkungen der Heparinoide werden in Abhhgigkeit des Sulfatiemngsgiades und
weitenx seukturellerParameterdiskutiert.
2. Galactomunnan
Galactomannans of the Guaran-type are known to consist
of a @1,4linked mannose backbone in which each third
C-6 position carries a galactose residue. After sulphatation”
a MW of 360 OOO D and a sulphur content of 3.1%, corresponding to a DS of 0.2, was obtained.
Derivatisationof polysaccharide mode{ structures
Ga I
@I6
1
1. Lichenin
B
B
Lichenin is known to be a linear, neutral ~-1,3/1,4-glucan
[ Manl--4Man1--4Man
with a ratio of 3:7 for p-1,3 and p-1,4-linkages respectiveFig. 2: Repeating unit of Galactomannan
ly?
B
1,
3. Arabinogalactan
B
[ G I C ~ ~ ~ G I C , - + ~ G In
LC
1,3 Glc : 1,4 Glc (3:7)
Fig. 1: Repeating unit of Lichenin
The third model polysaccharide was an acid and highly
branched arabinogalactan’). This polymer consists of a main
chain of p-1.6-linked galactose residues branched at C-3
Abbreviations:
M W molecular weight; DP degree of polymerisation; DS: degree of substitution: D Dalton: GPC gel permeation chromatography: A m . activated
partial thmmboplastin time; ‘IT thrombin time;axa: antifactor Xa activity; R1: refractometric index: Glc: glucose: Man: mannose; Gal: galactose; Ara:
arabinose;Gala: galacturonic acid.
+) H e m
Prof. Dr. Dr, E. Mutschler zum 60.Geburtstag zugeeignet.
Arch. Pharm. (Wcinheim) 324,363-365 (1991)
QVCH VerlagsgesellschaftmbH, D-6940Weinheim, 1991
0365-6233/91/0606-0363 $3.50 + .25B
Bode und Fnurz
364
with galactose residues. Arabinose residues are linked in the vatives was carried out in vitro with human pool- lasma by
and the a X a activity?I .
same way to the polysaccharide chain and are carrying a- evaluating the
Heparinoids were dissolved in 0.9% NaCl and tested in a
1.4-linked galacturonic acid residues.
After sulphatation*) a polymer with a M w of 23 OOO D concentration range of 1-200 pg/ml plasma against a blank
and sulphur content of 8.93% corresponding to a DS of and compared to the nonderivatized polysaccharides in a
similar concentration range. The three non-derivatized poly0.55, was obtained.
saccharides did not show any anticoagulant activity. The
B
B
B
B
B
-6Gall
-6Gal,
-6Gal,
-6Gall
-6Gall
-6Galldeviation of the clotting time of the aPlT and the 'IT is
about 5-7%, concerning aXa activity, there is a deviation of
about 24%.
The anticoagulant activity of lichenin is shown in Tab. 1:
GalA
Gal
Arc
Ara
Ara
Starting from a concentration of 5 pgiml plasma, this product
has a pronounced anticoagulant activity. Above 25 pg/ml
plasma an irreversible prolongation of both, the AF"IT and
Gal
Ara
Gal
the TF is taking place.
Compared to the first sulphated polymer, the derivatized
galactomannan proved to have much less anticoagulating
activity (Tab. 2):
Gal
Only at a concentration of 200 pgml plasma, a weak aXa
activity can be seen.
The highest anticoagulating activity was determined in the
Ara
case of the highly branched acidic and sulphated arabinogalactan. At concentrations as low as 5 pg/ml plasma, all antiFig. 3: Repeating unit of an acid Arabinogalactan
thrombotic parameters tested were shown to be strongly influenced
(Tab. 3).
Anticoagulant activity of different heparinoids
From these data it is obvious that the molecular structure
The testing method for the determination of an anticoagu- and composition, further the degree of sulphatation and the
lant activity of the newly synthesized polysaccharide deri- uronic acid content of a polymer are important parameters
.I:
4:
8: 4: 4: 4:
4:
4:
.I:
.i:
Tab. 1: Anticoagulant activity of the sulphated Lichenin
Anticoagulating
parameters
determined as:
(sec)
Heparinoid concentration (pglml plasma)
50
25
10
200
100
APTT
>240
Tr
>240
>240
>240
>240
>240
>240
105
45
18
14
ax,
>240
5
1
105
75
40
21
13
38.5
16.8
13.1
5
I
0
46
35
13
42
18
41.8
17.8
13
12.9
170
150
14
14
0
Tab. 2: Anticoagulant activity of the sulphated Galactomannan
Anticoagulating
parameters
determined as:
200
100
50
IT
240
240
240
240
ax,
53
26
222
240
15
Heparinoid concentration (&ml
25
10
plasma)
(W
m
64
140
161
14
65
I?
Tab. 3: Anticoagulant activity of the sulphated acid arabinogalactan
Anticoagulating
parameters
determined as:
(sec)
APlT
Tr
ax,
200
100
>240
>240
>240
>240
116
61
Heparinoid concentration (pg/ml plasma)
50
25
10
>240
>240
27
215
>240
16
132
130
14
5
1
0
19
29
13
42
IS
13
41.8
17.8
12.8
Arch. Pharm. (Weinheim) 324.363-365 (1991)
365
New Heparinoids from Plant Polysaccharides
for the anticoagulant activity. However, it is still uncertain if
the position of the SO3-groups at specific C-atoms is an
additional factor influencing the physiological activity of
heparinoids.
We are grateful for financial support by the ‘Fonds der Chemischen
Industrie’ and for a fellowship of the SANDOZ-foundation.
Experimental Part
1. Origine of the genuine polysaccharides: Lichenin: Rothchemicals;
Galactomannan: Serva-chemicals;ArabinogaIactan:obtained by the courtesy of Dr.1. Kraus (Regensburg).
2. GPC of the derivatized polymers was done in a Phannacia P 500LCC-500 system with a Superose TM 6 and a Superose Th4 12 column
(10x300 mm). 0.1 M NaCl solution served as the eluent at a flow rate of 30
mVh. Fractions were tested for polysaccharides by the anthrone method6)
and by the aid of an Erma-RIdetector FXC-7512.
The columns were calibratedwith standard pullulans (Mxbery and Nagel).
3. The derivatisation of the polysaccharides was carried out following
Karrer et at”: 0.2 g Lichenin were suspended in 5 ml pyridine and stirred
at I 10°C for 60 min with a mixture of chiomulphonic acidpyridine (l:7),
The reaction mixture was precipitated in acetone/methanol (91), washed
several times with acetone, dissolved in a small amount of water, dialyzed
for 48 h and freeze dried.
I n the case of glactomannan, the polymer (0.1 g) was suspended in 5 ml
pyridine at 65OC and treated as above for 2.5 h with the sulphating agent at
the same temp. Precipitation and purification was done as in the case of
lichenin.
The acidic arabinogalactan(50 mg) was derivatized following Lam and
Larsson”. The reaction mixture was taken to mom temp. and stirred for 2.5
h. 5 ml of pyridine were added and the reaction mixture was precipitated in
acetone/methanol (91). washed with acetone and recovered as in the case
of the derivatized lichenin.
4. For human plasma preparation venous blood was mixed with sodium
citrate 0.1 1 M (1:9), centrifuged for 10 min at 3000 g, and the supernatant
plasma was recovered. The plasma was stored at -20°C.
Arch. Pharm. (Weinheim) 324.363-365 (1991)
5. The anticoagulant tests were carried out with the aid of a Coagulometer KC 10 (Amelung) at 37.8OC.
6.APTT was measured with 100 pl plasma containing 100 pI PathromtinR for 120 sec at 37°C. After the addition of 100 11 of a CaClz solution
(0.025 mol/l), the coagulation time was determined.
7. TT was determined by mixing 100 pI of plasma with 100 pl diethylbarbituric acidacetate buffer pH 7.6 at 37OC for 60 sec. After addition
of 100 pl Test=fhrombinR(31 pghnl) the coagulating time was determined.
8. &-activity was measured with the HeptestR-methodutilizing 100 pI
plasma and 100 PI factor X.The mixture was incubated for 120 sec at
37°C. After addition of 100 pl of a 1: 1 mixture of CaCIdcephaelinsulution
the coagulatingtime was measured.
9. Methylation of the sulphated polysaccharides was done according to
Hakonwr?) and Harriset al?’.
10. GC-MSanalysis of the methylated sugar derivatives: Hewlett Packard GC 5890 A, MSD 5790 systeme on a DB 1701-60N (0.25 mm/30 cm)
column (I and W Scientific).
11. Determination of the sulphur content: ‘Institut fiir Biochemie,
UniversiCit Stuttgart’, FRG.
References
D. Bergquist and 1. Nilson, Thromb. Res. 23,304 (1983).
A. Perlin and S. Suzuki, Can.J. Chem. 40.50 (1%2).
S.Hakomori, Biochem. J. 55,205 (1964).
DJ. Harris, R.J. Henry, A.B. Blakeny, and R.A. Stone, Carbohydr.
Res. 127.59 (1984).
5 J. Kraus, Dissertation Regensburg 1987.
6 D.L. Moms, Science 107,254 (1948).
7 P. K m r , H. Koenig, and E. Usteri, Helv. Chim. Acta 26, 1296
(1943).
8 0.Larm and U. Larsson, Carbohydr. Res. 73,332 (1979).
9 H.C. Godal, Thromb. Res. 33.77 (1974).
10 E. Hiller, Dtsch. Med. Wschr. 98,1367 (1973).
11 J. Harenberg, Ch. Giese. A. KniMler, and R. Zimmermann, Arztl. Labor32, 181 (1986).
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