THROMBOLYTIC ACTIVITY OF GANDHAGA KARUPPU – IN VITRO STUDY

A.Samsul Nisha Fathima^1*, Dr.G.Essakky Pandian M.D (s) ²

​1.PGscholar, Department of Gunapadam,

​2.Reader, Department of Gunapadam.

Government Siddha Medical College Palayamkottai, Tirunelveli, Tamilnadu, India

ABSTRACT:

BACKGROUND:

Cerebral Infarction is the major cause of Hemiplegia. It occurs due to thrombosis and embolism. Thrombolytic therapy also known as clot busting drug is used in the clinical area to treat Venous and Arterial thromboembolic events. Commonly used thrombolytics are Streptokinase, Urokinase, Tissue Plasminogen Activator to dissolve the clots. Since thrombolytic agents are not in affordable cost, I have started to look for an alternate Medicine. The present study is aimed to investigate the In vitro Thrombolytic activity of Gandhaga Karuppu in Healthy Human Blood Sample.

OBJECTIVES:

To Evaluate the Thrombolytic activity of Herbo-mineral formulation of “Gandhaga Karuppu”

MATERIALS AND METHODS:

The Ingredients used in the preparation of Nellikai Gandhagam (Sulphur), Chukku (Dried Ginger), Milagu (Black Pepper) and Thippili (Long Pepper). The trial drug was prepared as perSiddha Literature, Kannusamy Parambarai Vaithiyam Pg.No.442. The Thrombolytic activity of Gandhaga Karuppu was evaluated using Healthy Human Blood Sample.

RESULT:

The in vitro study revealed that Gandhaga Karuppu showed 32.70% of Thrombolytic activity.

CONCLUSION:

The Present study proposes that the test drug “Gandhaga Karuppu” has moderate thrombolytic activity. Thus the formulation may be a source of effective herbo-mineral Siddha formulation.

KEYWORDS: Thrombolytic activity, Thrombolytics, Gandhaga Karuppu.

INTRODUCTION:

Human beings possess inbuilt system by which the blood remains in the fluid state normally and guards against the hazards of thrombosis and haemorrhage. Virchow Triad described three primary events which predispose to thrombus formation viz; endothelial injury, altered blood flow and hypercoagulability of blood. Along with this, the activation of Platelets and Clotting System occur. Thrombus activates the fibrinolytic system with consequent release of Plasmin which may dissolve the thrombus completely result in resolution. Usually lysis is complete in small venous thrombi; while large thrombi may not be dissolved. If the thrombus is not removed, it starts getting organized due to more and more deposition from the constituents of flowing blood ultimately cause obstruction of some important blood vessels. Thrombus developed in the circulatory system due to failure of haemostasis causes vascular blockage and leads to serious consequences such as acute Myocardial Infarction and Stroke. Stroke and Heart Attacks are the major causes of morbidity and mortality in the developed countries. Thrombolytic agents used to dissolve the clot and in the management of thrombosis in patients. Thrombolytic agents such as Tissue Plasminogen activator, Urokinase and Streptokinase are used all over the world for the treatment but their use is associated with high risk of Haemorrhage, Anaphylactic reaction and lack of Specificity. Therefore, in recent years research is focused on Traditional herbal medicines which have antiplatelet, anticoagulant and thrombolytic activity. The present study has been aimed to evaluate the in vitro Thrombolytic activity of Gandhaga Karuppu.

MATERIALS AND METHODS:

Collection of Raw drugs:

Nellikkai Gandhagam was bought from Gopalan aasaan shop, Nagarkovil at Kanyakumari district, Tamilnadu.

Other raw drugs were bought from Vallalar naatu maruthu kadai, Tirunelveli Town, Tamilnadu

“Gandhaga Karuppu” is a herbo-mineral formulation has the ingredients of

METHOD OF PREPARATION:

The purified Chukku, Milagu, Thippili were made into a powder( chooranam) individually and filtered by using sieve(80-100 meshes). Half of the above powder is placed in a mud plate (Agal) and then the entire quantity of purified Gandhagam is placed over the above powder and then the remaining powder (chooranam) is spread over the Gandhagam. This mud plate (Agal) was covered with another suitable mud plate (Agal) and then sealed with clay soaked cloth and made into seven layers. The entire setup was subjected into incineration process by using 15 dried cow dung cakes. After cooling, the final product obtained is black in colour was collected and grind well. The prepared medicine was stored in a air tight container.

SHELF LIFE:

5 years.

DOSAGE:

Kundri edai – 130mg daily for 6 to 7 days.

ADJUVANT :

Honey, Ghee

INDICATIONS:

Fever, Jaundice, Hemiplegia, Muscle cramps, Radiating Pain, Megavayu, Fever with delirium and Breast Milk Congestion.

PATHIYAM:

Avoid Tamarind and sexual indulgence

REFERENCES:

Kannusamy Parambarai Vaithiyam Pg.No.442, Fifth Edition -2006.

PHARMACOLOGICAL ANALYSIS:

THROMBOLYTIC ACTIVITY OF GANDHAGA KARUPPU

Reagents and Chemicals:

Streptokinase (SK) vials of 15,00,000 I.U.10 blood (5ml) sample drawn from healthy human volunteers, Gandhaga Karuppu , Distilled Water.

Apparatus

Micro centrifuge tube (0.5ml/tube), Micropipette, Vortex mixer, 0.22-micron syringe filter, Beaker, Electric Balance, Incubator.

Experimental procedure:

Specimen:

Whole blood (5 ml) was drawn from healthy human volunteers (n=10) without a history of oral contraceptive or anticoagulant therapy. 500 μl of blood was transferred to each of the ten previously weighed alpine tubes to form clots.

Sample preparation:

100mg of test drug Gandhaga Karuppu was suspended in 10 ml distilled water and the suspension was shaken vigorously on a vortex mixer. Then the suspension was kept overnight and decanted to remove the soluble supernatant, which was filtered through a filter paper. The solution was then ready for in vitro evaluation of clot lysis activity.

Streptokinase (SK) solution preparation:

To the commercially available lyophilized SK vial (Polamin Werk GmbH, Herdecke, Germany) of 15,00,000 I.U., 5 ml sterile distilled water was added and mixed properly. This suspension was used as a stock from which 100μl (30,000I.U) was used for in vitro thrombolysis.

Thrombolytic assay:

Experiments for clot lysis were carried as reported earlier

(1). Venous blood drawn from healthy volunteers was transferred in different pre- weighed (A) sterile eppendorf tube (500μl/tube) and incubated at 37 °C for 45 minutes. After clot formation, serum was completely removed (aspirated out without disturbing the clot formed). Each tube having clot was again weighed (B) to determine the clot weight (clot weight(C) = weight of clot containing tube-weight of tube alone). Each Eppendorf tube containing clot was properly labelled and 100μl of aqueous extract Gandhaga Karuppu was added to the tubes. All the tubes were then incubated at 37°C for 90 minutes and observed for clot lysis. After incubation, fluid obtained was removed and tubes were again weighed (D) to observe the difference in weight after clot disruption. Difference obtained in weight (E) taken before and after clot lysis was expressed as percentage of clot lysis

(2). Streptokinase and water were used as positive and negative control, respectively. The experiment was repeated ten times with the blood samples of different ten volunteers.

% clot lysis = (Weight of the lysis clot /Weight of clot before lysis) × 100.

Results

Thrombolytic activity assay

Addition of 100μl SK, a positive control (15,00,000 I.U.) to the clots along with 90 minutes of incubation at 37°C, showed 72.26% clot lysis. Clots when treated with 100μl sterile distilled water (negative control) showed only negligible clot lysis (3.80%) and clot treated with Gandhaga Karuppu showed 32.72%. Statistical representation of the effective clot lysis percentage by our Siddha classical preparation, positive thrombolytic control(Streptokinase) and negative control(sterile distilled water) is tabulated in (Table 1)

Table:1 Average percentage of clot lysis effect of GK

Table No:2 Effect of GK on clot lysis formulation

Figure:1 Effect of GK on clot lysis formulation

Table No:3 Group statistics (Control Vs Streptokinase)

Table No:4 Group statistics (Control Vs Formulation(GK))

Table No:5 Group statistics (Formulation(GK) Vs Streptokinase)

Table No:6 Effect of clot lysis

Figure:2 Effect of clot lysis

DISCUSSION:

The present study was undertaken to evaluate the thrombolytic activity of Gandhaga Karuppu. In the thrombolytic bioassay result suggested that the Gandhaga Karuppu showed very significant activity. The Gandhaga Karuppu can be evaluated to further research for thrombolytic activity to a specific disease.

Atherosclerosis-induced heart attacks and strokes are leading reasons of morbidity and mortality. Current essential and auxiliary prevention strategies emphasize control of different atherosclerotic danger components, including smoking, hypertension, hypercholesterolemia, diabetes mellitus, weight, irritation, and homocysteine . Current pharmacological studies recommend remedial estimations of these natural preparations, including lowering of blood pressure and lipids, anti-oxidation, thrombolytic activity and the promotion of microcirculation. There is a requirement for more goal and scientific approaches to authenticate individual herbs to identify chemical constituents, detect adulteration or contamination of herbs, and screen the quality of herbs and herbal medicines. There is also a need to check the consistency of different batches of herbs utilized as a part of this study and to distinguish bioactive parts in herbs reported to have physiological effects.

CONCLUSION:

Under this study, Test drug Gandhaga Karuppu of demonstrated moderate (P <0.001) clot lytic properties in different blood samples. The percent clot lytic activity was compared with water (Negative control) and standard enzyme streptokinase (positive control). The mean % of clot lysis for water and streptokinase was found to be 3.8% and 72% separately. The mean percent clot lytic activity of Test drug Gandhaga Karuppu was found to be 31.60%, which is significant compare with the positive and negative control. So, the present research proposes that, the Test drug Gandhaga Karuppu has moderate thrombolytic activity. Thus the formulation may be a source of effective herbo-mineral drug.

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