Cardiovascular disease and stroke are the major causes of morbidity and mortality in high-income countries. Although many factors contribute to the development of cardiovascular disease, thrombus formation is the main trigger event in acute coronary syndrome and stroke. Injury to the endothelial wall causes platelets to activate and adhere to the exposed endothelium, leading to thrombus formation [1].

Herbs and plants have been in use as a source of therapeutic compounds in traditional medicinal system since ancient time. Plant-produced compounds are of interest as sources of safer or more effective substitutes for synthetically produced antithrombotic agents. Herbal medicines have been used from the earliest times to the present day. [2]

Calotropis species, belonging to the family of Asclepidaceae in plant kingdom, are the well known plants throughout the tropical world and they are native to the tropical and subtropical parts of Asia and Africa [3]. C. gigantea is a wasteland weed better known as milkweed, habitat of Asian countries that includes, India, Indonesia, Malaysia, Philippines, Thailand, Sri Lanka and China. Tribal people were using this plant parts to cure several illnesses such as toothache, ear-ache, sprain, anxiety, pain, epilepsy, diarrhoea and mental disorders [4]. C. gigantea is scientifically reported for its anti-Candida activity, cytotoxic activity, antipyretic activity and wound healing activity.

Figure 1: Calotropis gigantea leaves

Phytochemicals present in the various parts of C. gigantea especially in the leaves are usharin, gigantin, alpha and beta-calotropeol, beta-amyrin, fatty acids (both saturated and unsaturated), hydrocarbons, acetates and the benzoates, a mixture of tetracyclic triterpene compounds, sterols, giganteol and giganteol are also found to be present. However cardenolide calotropin, α-amyrin, β-amyrin, β-sitosterol, α-amyrin methylbutazone, β-amyrin methylbutazone, α-amyrin acetate, β-amyrin acetate, lupeol acetate B, gigantursenyl acetate A, gigantursenyl acetate B, flavonol glycoside, akundarol, uscharidin, calotropin, frugoside, calotroposides A to G are responsible for many of its activities [5]. Current study was focused to investigate antithrombotic activity of aqueous and alcoholic extract of C. gigantea leaves.

MATERIALS AND METHODS:

Plant material:

C. gigantea plant was collected from the natural population growing in the Vilad ghat, Ahmednagar, India during December 2014. The plant material was identified at the field using standard keys and descriptions.

Processing of the plant:

Plant leaves were collected washed and were shade dried at room temperature. Dried leaves were uniformly grinded using mechanical grinder. Ten gram of powdered leaves was soaked in 100 ml of distilled water and alcohol separately in a conical flask and loaded on an orbit shaker at a speed of 120 rpm for 24 hours. The mixture was filtered using Whatman filter paper number 1. The filtrate was concentrated using rotary vacuum evaporator and dried. Dried aqueous and alcoholic extracts were collected in an air tight container and stored at 4°C. The aqueous and alcoholic extracts were dissolved in sterilized distilled water separately to make 1000 μg/ml stock solution and used to perform antithrombotic assay [1].

Determination of Prothrombin Time (PT) by Quick‘s Method:

Pipette out 0.1 ml of plasma in small test tube. Add 0.1 ml of brain thromboplastin and mix. Wait for 2 min and add pre warmed calcium chloride solution at 37°C, mix Start the stop watch. Hold the tube in front a source of light and keep tilting the test tube gently. At first appearance of fibrin clot stop the watch immediately and record the time as control reading. Pipette 0.1 ml of plasma in small test tube; add 0.1 ml of 100 mg/ml test compound solution. Incubate for 5 min and repeat the procedure to record the elongation in mean prothrombin time. [6]

RESULTS AND DISCUSSION:

The aqueous and ethanolic extracts of C. gigantea were subjected to preliminary phytochemicals screening and revealed the presence of Alkaloids, Phenolic compounds/tannins and flavonoids and Sterols. Anti-coagulant activities of aqueous and ethanolic extracts of C. gigantea were carried out by Quick’s method and results are shown in Table 1 and Table 2. The clot lysis of C. gigantea was found to be increased with the increase in concentration of the sample. Result shows that the extracts exhibit moderate anti-coagulant activity. Literature shows that tannins and flavonoids are responsible for anti-coagulation [7-8]. Hence the presence of tannin and flavonoid may contribute the said activity. Further study is under progress to isolate the pure component fraction.

Table no. 1: In-vitro anti-coagulant activity of ethanolic extract of Calotropis gigantia leaves

Sr. No.	Concentration (mg per dl)	Time (In sec.)			Mean
1.	100	42	41	40	41.00
2.	250	75	72	77	74.66
3.	500	180	182	180	180.66

Table no. 2: In-vitro anti-coagulant activity of aqueous extract of Calotropis gigantia leaves

Sr. No.	Concentration (mg per dl)	Time (In sec.)			Mean
1.	100	35	35	38	36.00
2.	250	70	70	70	70.33
3.	500	167	160	168	165.00

Table no. 3: In-vitro anti-coagulant activity of Warfarin

Sr. No.

Concentration

(mg per dl)

Time (In sec.)

Mean

100

102

103

102.66

CONCLUSION:

From the present study it is evident that aqueous and ethanolic extract of C. gigantea possess anticoagulant property. However, in vivo activity of C.gigantea is yet to be discovered and is in pipeline. By above result, it can be suggested that the application of the C. gigantea component may be accessible to the society for the treatment of cardiovascular diseases.

REFERENCES:

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2. Aarti C. A review on pharmacological and biological properties of Calotropis gigantea. International Journal of Recent Scientific Research. 5 (4); 2014: 716-719.

3. Singh S, Singh Sanchita and Singh AP. Phytochemical investigation of different plant parts of Calotropis gigantea. International Journal of Scientific and Research Publications. 3 (9); 2013; 1-3.

4. Kumar G, Karthik L and Rao KVB. Antibacterial activity of aqueous extract of Calotropis gigantea leaves – An in vitro study. International Journal of Pharmaceutical Sciences Review and Research. 4 (2); 2010: 141-144.

5. Kumar PS, Suresh E and Kalavathy S. Review on a potential herb Calotropis gigantea (L.) R. Br. Scholars Academic Journal of Pharmacy. 2 (2); 2013:135-143.

6. Sawant RL, Mhaske MS and Wadekar JB. Anticoagulant potential of schiff bases of 1,3-oxazines, International Journal of Pharmacy and Pharmaceutical Sciences. 4 (4); 2012: 320-323.

7. Umesh MK, Sanjeevkumar CB, Nayaka HB and Londonkar RL. Evaluation of in vitro anti-thrombolytic activity and cytotoxicity potential of Typha angustifolia L leaves extracts. International Journal of Pharmacy and Pharmaceutical Sciences. 6 (5); 2014: 81-85.

8. Vipin PS, Johannah NM, Menon S , Lawrence L , Raghavamenon AC and Padikkala J. Antithrombotic and anticoagulant activities of Desmodium gyrans (DC). Journal of Chemical and Pharmaceutical Research. 7 (5); 2015: 973-980.

Received on 20.01.2016 Modified on 25.01.2016

Research J. Pharm. and Tech 2016; 9(9):1493-1495.

DOI: 10.5958/0974-360X.2016.00290.0