Chapter 9 : Thrombolytic activity

Chapter 9 : Thrombolytic activity

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 Chapter 9 : Thrombolytic activity

9. In vitro thrombolytic activity of Methanolic extracts of leaves of Grewia nervosa

through in vitro clot lysis model

9.1. Introduction: Thrombolytic drugs are widely used for the management of cerebral venous
sinus thrombosis patients. Thrombolytic drugs like tissue plasminogen activator (t-PA),
urokinase, streptokinase etc. play a crucial role in the management of patients with CVST. In the
subcontinent though streptokinase and urokinase are widely used due to its lower cost, as
compared to other thrombolytic drugs, these are dangerous (Patel et al., 1999; Haines, 1995)
because they might cause serious bleeding complications along with reocclusion and
reinfarction. So search for alternative thrombolytic agents from plant source might be beneficial.
Since leaves of Grewia nervosa contain a variety of water soluble phytoconstituents, it is
possible that it may affect thrombolysis. Thus, the aim of this study was to examine the
thrombolytic potential of methanolic extract of leaves of the plant by using In Vitro clot lysis
model described by Prasad et al., 2006 and Ratnasooriyaet al., 2008.

9. 2. Materials and Methods

9.2.1. Preparation of sample:

600 mg of crude methanolic extract of roots of the plant was taken in a volumetric flask and a
stock solution of 20 mg/mL was made using 0.9% NaCl, the final volume being 30 ml. The
prepared stock solution was used to make different concentrations of root extract in isotonic
saline: 2.5, 5, 10 and 20 mg/mL.

9.2.2. Specimen
Venous blood was drawn from healthy human volunteers (n = 10) without a history of oral
contraceptive or anticoagulant therapy .A consent form was filled up for every volunteers before
collecting the blood.500 μl of blood was transferred to each of the previously weighed micro
centrifuge tubes to form clots.

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 Chapter 9 : Thrombolytic activity

9.2.3. Streptokinase (SK)

To the commercially available lyophilized streptokinase (15, 00,000 I.U.) vial (Eptase, Beacon
Pharmaceuticals Ltd, Bangladesh). 5 ml phosphate buffered saline (PBS) was added and mixed
properly. The conc. of the streptokinase became 30,000 I.U. which was used as the reference
standard for thrombolytic activity.

9.3 Study design

Venous blood drawn from healthy volunteers (n = 10) was immediately citrated using 3.1%
sodium citrate solution8 and then was transferred in different pre weighed sterile microcentrifuge
tube (500 μl/tube). Two hundred microlitres of 2% calcium chloride was then added to each of
these tubes4, mixed well and incubated at 37°C for 45 minutes for clotting to occur. After clot
formation, serum was completely removed (aspirated out without disturbing the clot formed) and
each tube having clot was again weighed to determine the clot weight (clot weight = weight of
clot containing tube – weight of tube alone). Each microcentrifuge tube containing clot was
properly labeled and five hundred microlitres of different concentrations of the plant extract, 2.5
mg/mL (n = 10), 5 mg/mL (n = 10), 10 mg/mL (n = 10) and 20 mg/mL (n = 10) or
saline(negative control)(n = 10) or 30,000 I.U. of streptokinase [ (Eptase, the Beacon
Pharmaceuticals Ltd , Bangladesh), reference drug (n = 13)] was added to tubes with clots. All
the tubes were incubated at 37ºC for 90 min. The fluid left was then carefully removed and the
tubes were weighed again. The difference in weight before and after clot lysis was expressed as
% clot lysis..

9.4 Statistical Analysis

The results are expressed as mean ± SEM. Statistical comparisons were made using one-way
ANOVA with Dunnett t test. Significance was set at p < 0.05. Dose dependencies were
determined by the regression coefficient (r).

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 Chapter 9 : Thrombolytic activity

9.5 Result and Discussion

A clear, visual representation of clot lysis is shown in figure no. 5.26. When 500 microlitre of
normal saline was added to the control clot minute clot lysis was observed. Whereas, tubes to
which streptokinase and different dilutions of plant extracts was added, significant clot lysis
could be visually seen. Percent clot lysis obtained after treating clots with streptokinase, control
and plant extracts group is shown in figure 2. The mean clot lysis % of plant extracts with all
different concentration was found to differ significantly when compared with control (i.e, p <
0.001 for all the concentrations).

Figure 9.1: Clot-lysis of blood samples of normal subjects (positive and negative control).

Tube no. 1 is a control clot (negative control) to which normal saline was added. A minute clot
lysis was observed (7.41%) in tube no.1; marked by the intact clot. The middle tube (positive
control) was lysed by 30,000 I.U. of streptokinase .After dissolution of the clots, tubes were
inverted and fluid along with the remnants of clots could be clearly seen. The right handed tube
was lysed by 20 mg/ml of leaves extract and 40.894% clot lysis was observed while that of
streptokinase was 48.91%4.

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 Chapter 9 : Thrombolytic activity

Table 9.1(a) : Effect of Streptokonase& normal saline on blood clot lysis of human blood in
vitro

SL no SIN W1 W2 W3= W4 W5= W2- % 0f clot Average %

W2-W1 W4 lysis of clot lysis
1 Con 1
1.031 1.291 0.26 1.246 0.045 17.30769231
2 Con 2
0.807 1.074 0.267 1.016 0.058 21.72284644
3 Con 3 1.025 1.295 0.27 1.28 0.015 5.555555556
4 Con 4
1.033 1.309 0.276 1.234 0.075 27.17391304
5 Con 5
0.835 1.033 0.198 1.015 0.018 9.090909091 15.90098411
6 Con6
1.025 1.272 0.247 1.215 0.057 23.07692308
7 Con 7
0.773 1.003 0.23 0.975 0.028 12.17391304
8 Con8
1.03 1.34 0.31 1.284 0.056 18.06451613
9 Con 9
0.787 1.002 0.215 0.978 0.024 11.1627907
10 Con 10
0.811 1.118 0.307 1.076 0.042 13.68078176
11 Std 1 1.023 1.29 0.267 1.195 0.095 31.74
12 Std 2 0.801 1.11 0.309 0.953 0.157 50.12
13 Std 3 1.019 1.345 0.326 1.171 0.174 53.5
14 Std 4 1.021 1.307 0.286 1.18 0.127 44.9 48.91
15 Std 5 0.83 1.093 0.263 0.932 0.161 62.2
16 Std 6 0.768 0.999 0.231 0.923 0.076 30.82
17 Std 7 1.031 1.275 0.244 1.155 0.12 49.23
18 Std 8 1.025 1.344 0.319 1.172 0.172 55.1
19 Std 9 1.029 1.345 0.316 1.204 0.141 44.6
20 Std 10 1.022 1.38 0.358 1.154 0.22 63.9
*SIN=Sample identification number, W1=Initial weight of eppendrof tube, W2= Weight after clot
formation; W3=Weight of Clot; W4=Weight after lysis of clot after application of Sample, w5= amount
of released clot ;stdt=Standard; 10 volunteer serial 1 to 10. All weight measured in mg.

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 Chapter 9 : Thrombolytic activity

Table 9.1(b): Effect of methanolic extract of leaves of G.nervosa on blood clot lysis of
human blood in vitro.

SL no SIN W1 W2 W3= W4 W5= W2- % 0f clot Average %

W2-W1 W4 lysis of clot lysis

21 Cr 20
0.804 1.038 0.234 0.987 0.051 21.79487179
22 Cr 20
1.024 1.46 0.436 1.297 0.163 37.3853211
23 Cr 20
1.035 1.338 0.303 1.254 0.084 27.72277228
24 Cr 20
1.033 1.31 0.277 1.241 0.069 24.90974729
25 Cr 20
0.811 1.257 0.446 1.017 0.24 53.81165919 40.89459088
26 Cr 20
0.8 1.45 0.65 1.009 0.441 67.84615385
27 Cr 20
0.798 1.159 0.361 0.981 0.178 49.30747922
28 Cr 20
1.027 1.452 0.425 1.262 0.19 44.70588235
29 Cr 20
1.027 1.439 0.412 1.25 0.189 45.87378641
30 Cr 20
1.022 1.362 0.34 1.241 0.121 35.58823529
31 Cr 10
0.8 1.223 0.423 0.989 0.234 55.31914894
32 Cr 10
1.021 1.446 0.425 1.268 0.178 41.88235294
33 Cr 10
0.797 1.121 0.324 1.027 0.094 29.01234568
34 Cr 10
0.836 1.277 0.441 1.021 0.256 58.04988662
35 Cr 10
1.027 1.299 0.272 1.226 0.073 26.83823529 32.99159848
36 Cr 10
1.022 1.251 0.229 1.205 0.046 20.08733624
37 Cr 10
0.771 1.041 0.27 0.952 0.089 32.96296296
38 Cr 10
0.801 1.091 0.29 1.034 0.057 19.65517241
39 Cr 10
1.024 1.314 0.29 1.263 0.051 17.5862069
40 Cr 10
1.021 1.312 0.291 1.229 0.083 28.52233677
*SIN=Sample identification number, W1=Initial weight of eppendrof tube, W2= Weight after clot
formation; W3=Weight of Clot; W4=Weight after lysis of clot after application of Sample, w5= amount
of released clot ;stdt=Standard; 10 volunteer serial 1 to 10. All weight measured in mg.

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 Chapter 9 : Thrombolytic activity

Table 9.1 (c): Effect of methanolic extract of leaves of G.nervosa on blood clot lysis of
human blood in vitro.

SL no SIN W1 W2 W3= W4 W5= W2- % 0f clot Average %

W2-W1 W4 lysis of clot lysis
41 Cr 5
0.813 1.053 0.24 0.997 0.056 23.33333333
42 Cr 5
0.804 1.132 0.328 1.02 0.112 34.14634146
43 Cr 5 0.782 1.129 0.347 1.006 0.123 35.44668588
44 Cr 5
1.03 1.332 0.302 1.228 0.104 34.43708609
45 Cr 5 28.02105133
1.027 1.258 0.231 1.194 0.064 27.70562771
46 Cr 5
0.801 0.981 0.18 0.945 0.036 20
47 Cr 5
1.025 1.306 0.281 1.226 0.08 28.46975089
48 Cr 5
1.03 1.351 0.321 1.262 0.089 27.7258567
49 Cr 5
0.806 1.078 0.272 1.031 0.047 17.27941176
50 Cr 5
0.802 1.068 0.266 0.992 0.076 28.57142857
51 Cr 2.5
1.02 1.331 0.311 1.205 0.126 40.51446945
52 Cr 2.5
1.029 1.324 0.295 1.252 0.072 24.40677966
53 Cr 2.5
1.029 1.327 0.298 1.247 0.08 26.84563758
54 Cr 2.5
1.025 1.291 0.266 1.214 0.077 28.94736842
55 Cr 2.5
0.792 1.015 0.223 0.953 0.062 27.80269058 27.71155224
56 Cr 2.5
1.021 1.267 0.246 1.195 0.072 29.26829268
57 Cr 2.5
0.802 1.081 0.279 0.969 0.112 40.14336918
58 Cr 2.5
1.021 1.372 0.351 1.27 0.102 29.05982906
59 Cr 2.5
1.033 1.295 0.262 1.254 0.041 15.64885496
60 Cr 2.5
0.821 1.06 0.239 1.018 0.042 17.57322176
*SIN=Sample identification number, W1=Initial weight of eppendrof tube, W2= Weight after clot
formation; W3=Weight of Clot; W4=Weight after lysis of clot after application of Sample, w5= amount
of released clot ; stdt = Standard; 10 volunteer serial 1 to 10. All weight measured in mg.

Table 9.2 : Effect of metanolic crude extracts of Leaves of G.nervosa on blood clot lysis of
human blood in vitro (mean± SEM)

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 Chapter 9 : Thrombolytic activity

Concentrations of plant Number of % Blood clot lysis

extracts, control and
population
standard
(n)
0.9% Nacl Solution 10 15.90 ± 2.14

Streptokinase (30,000, I.U) 10 48.91 ± 3.12***

MEL20 mg/ml 10 40..89 ± 4.51***
MEL 10 mg/ml 10 32.99 ± 4.54**

MEL 5 mg/ml 10 28.02 ± 2.54

MEL 2.5 mg/ml 10 27.71 ±1.93
***p<0.001, **p<0.01and *p<0.05 compared to control, one-way ANOVA with Dunnett t test
MEL=Methanolic extract of leaves

% of Clot lysis
60
48.71
50
40.89
40 32.99
28.02 27.71
30
20 15.9 % of Clot lysis
10
0
l l l
ro ** ** l*
* m m
on
t d* l* m g/ g/
C d ar g /m g/ 5m .5m
an m 0m n 2
St 0 1 Co n
n
2
on Co
Co C

Figure 9.2 : Clot lysis of blood samples of normal subjects by different concentrations of
Methanolic leaves extract of G.nervosa

In the evaluation of thrombolytic activity by in vitro clot lysis model, the methanolic extract induced
significant (P< 0.001) clotlysis of human blood with 40.89 % at 20 mg/ml compared to control
(normal saline) (Table 7.2 ). The clots were treated by different concentrations of methanolic leaves
extract (20, 10, 5 and 2. 5 mg/ml respectively). At the dose of 10 mg/ml methanolic extract show

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 Chapter 9 : Thrombolytic activity

moderate thrombolytic activity (P<0.01) while the standard thrombolytic drug streptokinase (30000
I.U.) showed 48.91 % clot lysis. (Table 9.2)

9.6. Discussion

This study examined the thrombolytic potential of methanolic extract of G.nervosa in vitro clot
lysis model using human blood. The test model used is a newly developed cheap and simple
technique which can be performed with limited facilities available in countries like Bangladesh.
But this technique is validated, sensitive and reliable. The results show, for the first time, that
methanolic extract of G.nervosa possesses thrombolytic activity.
This is an important finding which may have important implications in cardiovascular health. In
addition, this finding may indicate the possibility of developing novel thrombolytic agents from
the plant.
The response is dose dependent as 20 mg/ml shows higher thrombolytic activity than 10 mg /ml,
10 mg/ml shows higher activity than 5 mg/ml and 5 mg/ml shows greater activity than 2.5mg/ml
(Fig.9.2).

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