INTRODUCTION:

A blood clot (thrombus) developed in the circulatory system due to the failure of hemostasis causes vascular blockage and while recovering leads to serious consequences in atherothrombotic diseases such as acute myocardial or cerebral infarction, at times leading to death. Commonly used thrombolytic agents are alteplase, anistreplase, streptokinase, urokinase and tissue plasminogen activator (TPA) to dissolve clots¹. All available thrombolytic agents still have significant shortcomings, including the need for large doses to be maximally effective, limited fibrin specificity and bleeding tendency. Because of the shortcomings of the available thrombolytic drugs, attempts are underway to develop improved recombinant variants of these drugs ². Heparin and Aspirin are only moderately efficient for acceleration of lysis and prevention of reocclusion, but are safe.

More selective thrombin inhibitors and antiplatelet agents are m ore potent, but their safety remains to be confirmed. Continued investigation in this area will provide new insights and promote progress toward the development of the ideal thrombolytic therapy, characterized by maximized stable coronary arterial thrombolysis with minimal bleeding1. Several third generation thrombolytic agents have been developed. Compared with the second generation agents (altreplase), third generation thrombolytic agents such as monoteplase, tenecteplase, reteplase, lanoteplase, pamiteplase, and staphylokinase result in a greater angiographic potency rate in patients with acute myocardial infarction, although, thus far, mortality rates have been similar for those few drugs that have been studied in large -scale trials. Bleeding risk, however, may be greater³. Recently, preventive measures against thrombosis have been tried. Oral administration of the fibrinolytic enzyme nattokinase was one example, which has been reported to enhance fibrinolytic activity in plasma and the production of tPA ⁴. Since ancient times, herbal preparations have been used for the treatment of several diseases. Herbal products are often perceived as safe because they are "natural"⁵. Epidemiologic studies have provided evidence that foods with experimentally proved anti-thrombotic effect could reduce risk of thrombosis. Herbs showing thrombolytic activity have been studied and some significant observations have been reported⁶. Advances of Phytochemistry and identification of plant compounds which are effective in curing certain diseases has renewed the herbal medicines. Herbs and their components possessing anti-thrombotic activity has been reported before; however herbs that could be used for thrombolysis has to be reported so far⁷.

The genus Bougainvillea in the Nyctaginaceae (4 O’ clock) family of plants has 18 species, with three that are horticulturally important Bougainvillea spectabilis, B. glabra and B. peruviana. Bougainvillea glabra ‘Snow White’ is a cultivar of the B. glabra ‘Choicy’ which has white bracts with the greenish veins⁸. Bougainvillea glabra ‘Choicy’ have been used by the traditional practitioner of Mandsaur in variety of disorders like diarrhoea, reduce stomach acidity, cough and sore throught, decoction of dried flowers for blood vessels and leucorrohea and decoction of the stem in hepatitis. The main part used is leaves. The reported constituents in leaf of Bougainvillea glabra ‘Choicy’ are alkaloids, flavonoids, tannins, saponins and proteins. The leaves of Bougainvillea glabra ‘Choicy’ are reported to have insecticidal activity, anti-inflammatory, anti-diarrhoeal activity, anti hyperglycaemic activity, anti-ulcer and anti-microbial activity⁹. In spite the numerous uses and pharmacological activity attributed of Bougainvillea glabra Choicy but no pharmacological information regarding the leaves of this plant cultivar Bougainvillea glabra ‘Snow White’. Their fore objective of present study was to evaluate thrombolytic activity of Bougainvillea glabra Linn leaves extract.

MATERIALS AND METHODS:

Collection of Materials:

Fresh leaves were collected from the Medicinal plants garden established at the green house of Anurag Group of Institutions, Kodad campus, during the month of July 2012. The leaves were identified by Department of Pharmacognosy.

Preparation of plant extract:

Collected plant material were shade dried and pulverized into coarse powder using mortar and pestle. The dried powdered material was extracted with methanol using Soxhlet apparatus and filtered using Whatman No. 40 filter paper. The extract obtained from the solvent were evaporated to dryness at room temperature and suspended in sterile distilled water to make the final concentration of 20mg/ml.

In vitro Thrombolysis:

Whole blood (n=10) were collected from the healthy volunteers without a history of oral contraceptive or anticoagulant therapy. For each treatment ten tubes were taken and experiment was repeated thrice.

Experiment 1: Effect of flower extract on clot lysis:

Blood sample (500µl) was distributed in pre weighed sterile micro centrifuge tubes and incubated at 37⁰C for 90min for clot formation. After clot formation, the serum was completely aspirated without disturbing the clot and the tubes were again weighed to determine the clot weight (clot weight = Weight of the tube containing clot – Weight of the empty tube). To the each Eppendorf tube containing preweighed clot, 200µl, 400 µl and 800 µl of methanol extract were added. For negative control, 500µl of sterile distilled water was used. All the tubes were incubated at 37⁰C for 18 hr and observed for clot lysis. The fluid obtained after the incubation was removed carefully and the tubes were weighed again to observe the difference in weight after clot disruption. Difference in the weight taken before and after clot lysis was expressed as percentage of clot lysis.

Experiment 2: standardization of concentration and incubation time for maximum clot lysis:

Various concentrations of leaf methanol extracts viz; 200µl,

400µl and 800µl were tested against various (24hrs, 48hrs, 72hrs and 96hrs) duration of incubation at 37⁰C for maximum clot lysis.

Statistical Analysis:

Data was analysed by one- way ANOVA by using student’s t-test. Significance was set at p<0.05.

RESULTS AND DISCUSSION:

Methanolic extract tested for clot lysis, proved its superiority maximum (94.5%) of clot lysis. Sterile distilled water as a negative control also showed negligible (3.83%) clot lysis. Concentrations of leaf methanol extract and incubation time were directly proportional to the clot lysis. Leaf methanol extract at the concentration of 800 µl for 72 hours exhibited 100% clot lysis (Table.1). Till now there is no report on the standardization of the herbal extract of Bougainvillea glabra for thrombolysis. Mechanism of blood clot lysis by the plant extract is very complex and clearly not known. The preliminary studies carried out for screening of phytochemicals from ethanol extract of leaf has shown the presence of phenolics, tannins, lignins, flavonoids, saponins, glycosides and terpenes, which may be leading to blood clot lysis. Clot lysis may be the result of the combinatorial effect of the active compounds present or by the individual compounds. With further research on cell viability tests and in vivo studies, this finding may have important implications in the treatment of cardiovascular diseases which is increasing at an alarming rate. Since the drugs used for the cardio vascular diseases are not economical and not accessible to the greater section of the society, application of this study may be a boon for them.

Table1: Effect of different concentrations of methanolic extract and its incubation duration on clot lysis

Concentration of extract	Incubation time	Average % clot lysis
200µg/ml	24	12.3
	48	32.6
	76	47.3
400µg/ml	24	25.2
	48	52.1
	76	67.4
800µg/ml	24	70.2
	48	86.3
	76	94.5
Streptokinase	24	84.4
	48	92.2
	76	98.5
Distilled water	24	10.4
	48	32.5
	76	52.4

ACKNOWLEDGEMENT:

Authors would like to thank the Management and Principal, Anurag Pharmacy College, Kodad.

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Received on 08.09.2012 Accepted on 22.10.2012

Asian J. Res. Pharm. Sci. 2(4): Oct.-Dec. 2012; Page 134-136