In vitro thrombolytic potential of root extracts of four medicinal plants available in Bangladesh.

CONTEXT: Thrombus formation inside the blood vessels obstructs blood flow through the circulatory system leading hypertension, stroke to the heart, anoxia, and so on. Thrombolytic drugs are widely used for the management of cerebral venous sinus thrombosis patients, but they have certain limitations. Medicinal plants and their components possessing antithrombotic activity have been reported before. However, plants that could be used for thrombolysis has not been reported so far. AIMS: This study's aim was to evaluate the thrombolytic potential of selected plants' root extracts. SETTINGS AND
DESIGN: Plants were collected, dried, powdered and extracted by methanol and then fractionated by n-hexane for getting the sample root extracts. Venous blood samples were drawn from 10 healthy volunteers for the purposes of investigation. SUBJECTS AND METHODS: An in vitro thrombolytic model was used to check the clot lysis potential of four n-hexane soluble roots extracts viz., Acacia nilotica, Justicia adhatoda, Azadirachta indica, and Lagerstroemia speciosa along with streptokinase as a positive control and saline water as a negative control. STATISTICAL ANALYSIS USED: Dunnett t-test analysis was performed using SPSS is a statistical analysis program developed by IBM Corporation, USA. on Windows.
RESULTS: Using an in vitro thrombolytic model, A. nilotica, L. speciosa, A. indica, and J. adhatoda at 5 mg extract/ml NaCl solution concentration showed 15.1%, 15.49%, 21.26%, and 19.63% clot lysis activity respectively. The reference streptokinase showed 47.21%, and 24.73% clot lysis for 30,000 IU and 15,000 IU concentrations, respectively whereas 0.9% normal saline showed 5.35% clot lysis.
CONCLUSIONS: The selected extracts of the plant roots possess marked thrombolytic properties that could lyse blood clots in vitro; however, in vivo clot dissolving properties and active components responsible for clot lysis are yet to be discovered.

INTRODUCTION

Like other developing countries, thromboembolic disorders are one of the main causes of morbidity and mortality in Bangladesh.[1] To dissolve clots commonly used thrombolytic agents are alteplase, anistreplase, streptokinase, urokinase, and tissue plasminogen activator.[2] All available thrombolytic agents still have significant shortcomings, including the need for large doses to be maximally effective, limited fibrin specificity, and bleeding tendency.

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.”[3] Epidemiologic studies have provided evidence that foods with experimentally proven anti-thrombotic effect could reduce the risk of thrombosis. Herbs showing thrombolytic activity have been studied, and a few significant observations have been reported.[4] Advances of phytochemistry and identification of plant compounds that are effective in curing certain diseases has renewed interest in herbal medicines. 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 bleeding.

Since the plants Acacia nilotica, Lagerstroemia speciosa, Azadirachta indica, and Justicia adhatoda contain a variety of water soluble phytoconstituents,[5678] it is possible that root extracts of these plants may affect thrombolysis. Thus, the aim of this study was to examine the thrombolytic potential of crude extract of roots of the selected four plants by using in vitro clot lysis model described by Prasad et al.[9] and Ratnasooriya et al.[10]

SUBJECTS AND METHODS

Plant collection and extraction

Roots of A. nilotica and A. indica were collected from Noakhali while J. adhatoda and L. speciosa were collected from Gazipur and Narail respectively and voucher specimens for each of the collections (DACB 38214, 38215, 38213, and 38212 respectively) were deposited in Bangladesh National Herbarium for future reference. The roots were first separated from the plants, cleaned, cut into small pieces and air-dried for several days.

The air-dried and powdered roots of the plants were separately soaked in methanol for 15 days at room temperature with occasional shaking and stirring. It was then filtered, and the filtrate was then reduced using a rotary evaporator at low temperature and pressure. The dried plant extracts were used for the experiment dissolving them in normal saline to get a solution of 5 mg/ml.

Blood specimen

Venous blood samples were drawn from 10 male healthy volunteers (age 22-24 years) without any recent history of oral contraceptive and anticoagulant therapy. About 500 μl of blood was taken into each pre-weighed microcentrifuge tube to form clots, and these were separated from each other by assigning a distinct number to each. A volunteer consent form and Ethics Committee approval letter were filed up for each volunteer for future reference.

Streptokinase

To the commercially available lyophilized streptokinase (15,00,000 IU) vial (S-Kinase, Popular Pharmaceuticals Ltd., Bangladesh) 5 ml phosphate buffered saline was added and mixed properly. The conc. of the streptokinase was adjusted to be 30,000 IU and 15,000 IU, which were used as the reference standard for thrombolytic activity since it is used as a common thrombolytic drug.[11]

Study design

Venous blood drawn from healthy volunteers (n = 10) was immediately transferred in different pre-weighed sterile micro-centrifuge tubes (500 μl blood/tube, 10 tubes for each plant extract). About 200 μl of 2% calcium chloride was then added to each of these tubes, mixed well and incubated at 37°C for 45 min 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 500 μl of plant extract, normal saline (as a negative control), 30,000 IU and 15,000 IU of reference streptokinase were added to tubes with clots. All the tubes were incubated at 37°C 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 percentage clot lysis.

Statistical analysis

The significance of percentage clot lysis between plants extracts and negative control (normal saline) by means of the weight difference was tested by the Dunnett t-test analysis. Significance were set at both P < 0.001 and P < 0.05 levels. Data are expressed as mean ± standard error mean. SPSS is a statistical analysis program developed by IBM Corporation, USA. was used to for this purpose.

Percentage clot lysis = (weight of the clot after lysis by sample and removal of serum/weight of the clot before lysis by sample) ×100.

RESULTS

Addition of 500 μl of streptokinase of 30,000 IU and 15,000 IU concentrations to tubes showed highly significant (P < 0.001) clot lysis of 47.21% and 24.73% respectively comparing with 5.35% clot lysis of normal saline considered as a negative control.

At 5 mg/ml concentration of root extract of A. nilotica, A. indica, and J. Adhatoda showed 15.1%, 21.26%, and 19.63% clot lysis activity respectively, which were highly significant (P < 0.001) comparing with negative control (normal saline). At same concentration, 15.49% clot lysis activity was found for L. speciosa that was significant (P < 0.05) compared with 5.35% clot lysis activity of normal saline [Table 1].

Table 1

Potentiality of n-hexane extracts of roots of ANRE, LSRE, AIRE and JARE on human blood clot lysis in vitro

DISCUSSION

The present results clearly show that saline water soluble root extract of selected plants have thrombolytic and/or fibrinolytic effect (s) as it reduces the clot weight. The results showed, for the first time, those roots of selected four plants possess thrombolytic activity.

The test model used is a newly developed, validated, sensitive, reliable, and simple technique[6] that can be performed with limited facilities available.

Various research works are undertaken in quest of thrombolytic drugs. More site specificity with fewer side effects of thrombolytic drugs are desirables in any natural thrombolytic product. Herbal drugs can be a source to address this concern. Few herbal medicines exert thrombolytic or fibrinolytic effects such as Fangonia arabica,[12] Artmisiae folium,[13] Hemidesmus indicus,[14] garlic,[15] and tea.[10]

This is an important finding, which may have implications in cardiovascular health especially in atherothrombotic patients. This is only a preliminary study and to make the final statement about the potentiality of these herbs as thrombolytic drugs may require further study. Studies may be undertaken to identify the chemical structure of the active ingredients of the root extracts and to elucidate the exact mechanism of action.