Evaluation of in vitro anti-thrombolytic activity and cytotoxicity potential of Typha angustifolia l leaves extracts

Abstract

Objective: The aim of this study was to investigate the in vitro thrombolytic activity and cytotoxicity of T angustifolia leaf extract. Methods: An in vitro thrombolytic model was used to evaluate the clot lysis effect of different extracts of T.angustifolia Linn along with Streptokinase as a positive control and distilled water as a negative control. The cytotoxic activity of diffetent extracts of T.angustifolia leaves was evaluated by Brine Shrimp Lethality Bioassay. Results: An in vitro thrombolytic model of aqueous, methanol and chloroform extracts have shown 51.76±2.5%, 58±2.32 and 18±1.84 clot lysis respectively, where as the positive control Streptokinase shown 79.6±1.10 and negative control shown negligible 2.44±0.62. The aqueous, methanol and chloroform extracts have shown brine shrimp lethality with LC50 value of 40μg/ml, 30μg and 104 μg/ml respectively. It was found that aqueous and methanol extracts of T angustifolia possesses potential thrombolytic activity as well as cytotoxicity. Conclusion: The present investigation revealed that the aqueous and methanol extracts of T angustifolia possesses thrombolytic properties as well as cytotoxicity effects and it can be further used for treatment of cardiovascular diseases and cancer.

Full text

Original Article
EVALUATION OF IN VITRO ANTI-THROMBOLYTIC ACTIVITY AND CYTOTOXICITY
POTENTIAL OF TYPHA ANGUSTIFOLIA L LEAVES EXTRACTS
UMESH M K
,
SANJEEVKUMAR C B, HANUMANTAPPA BHERIGI NAYAKA
AND RAMESH L LONDONKAR*
1
Department of Post Graduate Studies and Research in Biotechnology, Gulbarga University Gulbarga 585106, Karnataka, India
Email: londonkarramesh53@gmail.com
Received: 27 Jan 2014 Revised and Accepted: 25 Feb 2014
ABSTRACT
Objective: The aim of this study was to investigate the in vitro thrombolytic activity and cytotoxicity of T angustifolia leaf extract.
Methods: An in vitro thrombolytic model was used to evaluate the clot lysis effect of different extracts of T.angustifolia Linn along with
Streptokinase as a positive control and distilled water as a negative control. The cytotoxic activity of diffetent extracts of T.angustifolia leaves was
evaluated by Brine Shrimp Lethality Bioassay.
Results: An in vitro thrombolytic model of aqueous, methanol and chloroform extracts have shown 51.76±2.5%, 58±2.32 and 18±1.84 clot lysis
respectively, where as the positive control Streptokinase shown 79.6±1.10 and negative control shown negligible 2.44±0.62. The aqueous, methanol
and chloroform extracts have shown brine shrimp lethality with LC
50
value of 40µg/ml, 30μg and 104 µg/ml respectively. It was found that aqueous
and methanol extracts of T angustifolia possesses potential thrombolytic activity as well as cytotoxicity.
Conclusion: The present investigation revealed that the aqueous and methanol extracts of T angustifolia possesses thrombolytic properties as well
as cytotoxicity effects and it can be further used for treatment of cardiovascular diseases and cancer.
Keywords: Antithrombolytic, Brine Shrimp, Cytotoxicity, Typha Angustifolia.
INTRODUCTION
Nature had been known as stockyard of medicinal agents since the
time immemorial. Herbal products are extensively perceived as safe
because they are "natural"[1] having less or no side effects.
Medicinal plants contain large number of secondary metabolites
which have potential therapeutic properties that can be utilized in
the treatment of human diseases [2]. Primary bioassay screens are
most important for the initial screening of plants for bioactive
principles and are often the first step in drug development [3].
Medicinal plants have acquired significant importance in the field of
biotechnology for their developing applications [4]. Hence, in the
recent years the researchers are focusing on formulation of
ayurvedic herbal medicines on the basis of their traditional uses and
its known effectiveness in the treatment of various ailments.
Cardiovascular disease caused by blood clot (thrombus) formation is
one among the most severe diseases which are increasing at an
alarming rate in the recent years.[4] Thrombolytic agents are used to
dissolve clot and in the management of thrombosis in patients[5].
Thrombolytic agents such as tissue plasminogen activator (t-PA),
Urokinase (UK), streptokinase (SK)[6] etc, are used all over the world
for the treatment [7] but their use is associated with hyper risk of
haemorrhage [8], anaphylactic reaction and lacks specificity.
Remarkable efforts have been made towards the discovery and
development of natural constituents from various plant and animal
sources which have antiplatelet [9,10] anticoagulant [11,12],
antithrombotic [13] and thrombolytic activity [14-16]. The brine
shrimp leathelity bioassay was used as an indicator for general toxicity
and also as a guide for the detection of antitumor and pesticidal
compounds [17]. The brine shrimp lethality bioassay is efficient, rapid
and inexpensive tests that require in a relatively small amount
samples. The technique is easily mastered, costs little, and utilizes
small amount of test material[18]. Meyer et al.(1982)[19] have
successively studied for in-vivo lethality bioassay-guide fractionation
of active cytotoxic and antitumor agents such as trilobacin from the
bark of Asimina triloba [20], cis-annonacin from Annona muricata [21]
and ent- kaur-16-en-19-oic acid from Elaeoselinum foetidum [22].
Typha angustifolia of the family typhaceae is commonly known as
Elephant grass or cattail. This plant is characterized by its fast
growth and high biomass [23]. Several parts of the plant are edible,
including dormant sprouts on the roots and bases of the leaves, ripe
pollen, the stem and the starchy roots [24,25]. The traditional uses
of pollen grains of T. angustifolia for treatment of kidney stones,
abnormal uterine bleeding, abscesses, tapeworm infection diarrhea
and dysentery is well known [26]. Modern research on pollen grains
of angustifolia mainly reveals that it contain sterols, terpinoids,
flavonoid glycosides [27], cerebrosides and long chain hydrocarbons
that pocess various pharmacological activities like
immunosuppression[28], antiplatelet aggregation [29],
antimicrobial [30,2], cholesterol lowering activity and
antiatherogenic effect[31]. The rhizome flour of Typha angustifolia
used in the treatment of human IBD (Inflammatory bowel disease) is
also studied by Andréa et al (2012)[32]. The present study has been
designed to evaluate the antithrombolytic activity and cytotoxicity of
different extracts of T angustifolia Linn leaves.
MATERIALS AND METHODS
Collection and extraction of plant materials
Aerial part (leaves) of T .angustifolia was collected in and around
Gulbarga University campus, Gulbarga,Karnataka, India in the month
of March 2013. The collected plant materials were washed with
running tap water, allowed to air dry and were dried in shade for
two to four weeks. Precaution was taken to avoid direct sun light
contact of leaves otherwise it will destroy the active compounds of
plant leaves. After drying, the plant leaves were grinded finely and
stored in airtight container. The air dried leaf powders (50 g) were
successively extracted by soxhlet extraction with solvents of
increasing polarity i.e., petroleum ether (60°-80°C), chloroform,
methanol and distilled water. The extracts were dried and stored in
a sterile container for further use.
Clot lysis
The clot lysis was carried out as per the method reported by Prasad
et al 2006[33]. In brief, 2.5 ml of venous blood drawn from healthy
volunteers was distributed in 5 different pre weighed sterile
microcentrifuge tube (0.5 ml/tube) and incubated at 37°C for 45
minutes. After clot formation, serum was completely removed
without disturbing the clot and each tube having clot was again
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 6, Issue 5, 2014
Innovare
Academic Sciences

Londonkar et al.
Int J Pharm Pharm Sci, Vol 6, Issue 5, 81-85
82
weighed to determine the clot weight (clot weight = weight of clot
containing tube – weight of tube alone). To each microcentrifuge
tube containing pre-weighed clot, 100 μl of different extracts of
Typha angustifolis(Linn) is to be added. To the commercially
available lyophilized streptokinase vial (Lupiflo, Lupin Limited,
Mumbai, India) 2.5 ml of PBS was added and thoroughly mixed. This
suspension was used as a stock from which 100µl was added to the
microcentrifuge tube as a positive control. For negative control, 100
μl of distilled water were added. All the tubes were then incubated
at 37°C for 90 minutes and observed for clot lysis. After incubation,
the fluid released was removed and tubes were again weighed to
observe the difference in weight after clot disruption. Difference
obtained in weight taken before and after clot lysis was expressed as
percentage of clot lysis. The experiment was repeated 4 times with
the blood samples of 5 volunteers.
Brine Shrimp Lethality Bioassay
Brine shrimp cytotoxicity bioassay is very simple bench-top assay
used to measure cytotoxicity of plant extracts as well as synthetic
compounds [34, 35]. It was carried out with the method as described
by Meyer et al. (1982) [19] to investigate the cytotoxicity of the
extracts. The different extracts of Typha angustifolia were dissolved in
DMSO to obtain a stock solution of 10 mg/ml from which appropriate
(1-160µg/ml) dilutions were made to observe the cytotoxic activity.
Simple zoological organism (Artemia salina) was used as a convenient
monitor for cytotoxic screening. The commercially available eggs were
hatched in a small partitioned tank containing artificial seawater
(3.8% NaCl, pH 8.5) under constant aeration for 24h under the light
and allowed to grow further for 48 h to get shrimp larvae called
nauplii. With the help of Pasteur pipette add 10 brine shrimps to the
vial containing 5ml of artificial sea water. After 24 h, the vials were
inspected using a magnifying glass, and the number of survived nauplii
in each vial was counted. The mortality endpoint of this bioassay was
defined as the absence of controlled forward motion during 30 sec of
observation [19].
Statistical analysis
Results are expressed as Mean ± SEM. The statistical analysis was
carried out using one way ANOVA analysis. The p-value of 0.05 or
less was considered significant for all experiment.
Table 1: brine shrimp lethality tests of different extracts of T. angustifolia
S. No.
Conc in µg/ml
Aqueous Extract
Methanol Extract
Chloroform Extract
1.
1
13.33±5.77
17±5.77
3.33±5.77
2.
2.5
16.67±
5.77
20±10
6.67±5.77
3.
5
20±0
0
26.67±5.77
13.33±5.77
4.
10
23.33±5.77
36.67±
5.77
16.67±5.77
5.
20
36.67±
5.77
46.67±
5.77
26.67±
5.77
6.
40
50±0
0
53.33±
5.77
30±0
0
7.
80
56.67±11.5
63.33±
5.77
46.67±5.77
8.
160
73.33±15.3
86.67±
5.77
56.67±5.77
LC
50
40µg/ml
30µg/ml
104µg/ml
Values are expressed as Mean ± SEM, Sample volume 3(n=3), p<0.05.
Fig. 1: Dissolved clots after treating with different extracts of
T. angustifolia
RESULTS
The aqueous and methanol extracts showed 51.76±2.5%
and58±2.33% respectively where as chloroform extract shows
18±1.84%. Addition of 100 μl Streptokinase has showed 79.6 ±1.1%
clot lysis (Fig 1 & Fig 2). However, distilled water (negative control)
shown only negligible clot lysis (2.44 ± 0.62%). The mean difference
in clot lysis percentage between positive and negative control was
significant (p value < 0.0001). The mean percentage of clot lysis by
different extracts of T angustifolia was statistically more significant
Fig. 2: Percentage of Clot lysis by distilled water, Streptokinase
and different extracts of T. angutifolia
(p value < 0.0001) when compared to those of both positive control
streptokinase and negative control water.
In the brine shrimp test, among three extracts evaluated the
chloroform extracts having LC
50
> 100 µg/mL which is nontoxic or
exhibited weak toxicity. On the other hand, methanol (LC
50
30
µg/mL) and aqueous (LC
50
40 µg/mL) extracts have shown
significant toxic effects.
2.44%
79.60%
51.76%
58%
18%
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
% of Clot Lysis

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Int J Pharm Pharm Sci, Vol 6, Issue 5, 81-85
83
DISCUSSION
Medicines derived from plants origin will have a long history of use
for the prevention and treatment of various diseases. Approximately
30% of the pharmaceuticals formulations are prepared from plants
across the world[36] and are considered to be less toxic and freer
from side effects than the synthetic one [37]. Efforts have been
carried in recent past two decades towards the exploration,
discovery, designing and development of natural products with
antiplatelet[10], anticoagulant[12], antithrombotic [13] and
thrombolytic activity of the plants[38]. Few plant extracts and their
products having fibrinolytic activity are identified, which includes
Lumbricus rubellus [39], Pleurotus ostreatus [40], Spirodela polyrhiza
[41], Flammulina velutipes [42], and Ganoderma lucidum [43], Ginger
(Zingiber officinale) [44], Garlic (Allium sativum) [45] as well as from
Bacillus sp. in Korean and Japanese fermented foods, chungkook-
jang [46] and natto [47,48] respectively.
Generally blood clots are formed from fibrinogen by thrombin.
Antithrombotic or thrombolytic drugs can block the pathway of
thrombus formation. The fundamental task of thrombolytic therapy
is the degradation of fibrin by plasmin, which can be activated by the
activators from inactive plasminogen.[49]. T angustifolia is known
for antibacterial activity against six strains notably Enterobacter
aerogenes, Salmonella typhimurium, Klebsiella pneumonia,
Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus
aureus [2,30]. However, there are findings of bacterial contaminants
of plants which have plasminogen receptors that bind plasminogen.
Cell surface bound plasminogen is easily activated to plasmin, which
could lead to fibrinolysis [50]. Bacterial plasminogen activator:
staphylokinase, streptokinase, act as cofactor molecules that
contribute to exosite formation and enhance the substrate
presentation to the enzyme. Staphylokinase activates plasminogen
to dissolve clots, which also destroys the ECM and fibrin fibers that
hold cells together [51,52]. By comparing with this positive &
negative control, a significant thrombolytic activity was observed
after treating the clots with aqueous and methanol extract where as
chloroform extract of T angustifolia result indicates less potential to
lyse the clot. Thrombolytic activity (Clot lysis) of T angustifolia
extract may be the result of the combinatorial effect of the active
compounds present or by the individual compounds. Further
research on cell viability tests and in vivo studies, will have an
important implications in the treatment of cardiovascular diseases
which is increasing at an alarming rate. Since the commercially
available drugs used for the cardio vascular diseases are expensive
and not accessible to the greater section of the society, application of
this study may be a boon for them.
Herbal preparations will be a better option, if taken in an appropriate
dose for curing various ailments and if taken in higher/lethal dose
plant extracts could be harmful [53,54]. Toxicity of plant extract is a
major concern of scientists and medical practitioners. Several methods
of lethality tests have been successfully used to biomonitor the
isolation of cytotoxic, antimalarial, insecticidal and antifeedant
compounds from plant extracts [55]. Several lethality tests have been
designed and one such method is the lethality test wherein Brine
Shrimp Lethality (LC50, 24 hr.) test is used to determine cytotoxicity
of different plant extracts.
Brine shrimp lethality bioassay is a rapid and comprehensive
bioassay for the study of bioactive compound of the natural and
synthetic origin. Brine shrimp cytotoxic assay not only reveals the
cytotoxicity of the natural products and synthetic compounds but it
also supports anticancer, antiviral, insecticidal and pesticidal
potential [56]. This test is based on the potential effects of different
extracts of T angustifolia to become lethal to A. salina nauplii due to
its toxic expression. According to Meyer et al.(1982) [19], extracts
derived from natural products will have LC50 ≤ 1.0 mg/mL are
known to possess toxic effects. In this study, the table (Table 1 )
shows that the LC50 values of the aqueous, methanol and
chloroform extracts is 0.04, 0.03 and 0.104 mg/mL after 24 h,
respectively. Thus, these results prove that the aqueous and
methanol extracts of T angustifolia were significantly toxic
compared to chloroform extract. A good correlation has been found
between brine shrimp cytotoxicity and cytotoxicity against KB
(human nasopharingean carcinoma) cells [17]. Toxicity of the leaf
and seed extracts of Cassia alata by using brine shrimp cytotoxicity
assay was demonstrated by Awal et al., (2004) [57], where as
cytotoxic evaluation of components of Bolax gummifera was studied
by using brine shrimp cytotoxicity assay by Mongelli et al.,
(2003)[58]. While Chowdhury et al., (2004)[59] described that the
cytotoxic potential of extracts and purified components of
Stachytarpheta urticaefolia by using Brine shrimp assay.Hence, T
angustifolia leaf extract may be further explored for the
development of natural product-based pharmaceutical products.
CONCLUSION
Through our study it was found that aqueous and methanol extracts
of T angustifolia possesses thrombolytic properties as well as
cytotoxicity effects. However, in vivo clot dissolving properties and
active component(s) responsible for cytotoxicity of T angustifolia
are yet to be discovered. By the above obtained results, it can be
suggested that the application of the T.angustifolia component may
be accessible for greater section of the society for the treatment of
cardiovascular diseases and cancer.
CONFLIT OF INTREST
The authors declare that there is no conflit of interest.
ACKNOWLEDGEMENT
The author (Umesh M K) acknowledges to University Grant
Commission New Delhi (India) for providing financial support to
carry out this research. The Authors also thankful to Gulbarga
University Gulbarga, Karnataka (India) for providing lab facility to
carry out this study
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