A Preliminary In-Vitro Anticoagulant Activity of Karamarda(Carissa carandus L.)

Introduction

Carissa congesta Wight (syn. Carissa carandas Auct., C. carandas L.), belongs to Apocynaceae,¹ the dogbane family, and is found to be widely distributed throughout India. The shrub is commonly known as karonda, karamardaka (Sanskrit), koromcha (Bengali), vakkay (Telugu), kilaakkaai (Tamil), and Karja tenga (Asam). Fruits are berries and are commonly used as a condiment in Indian pickles.² The unripe fruits of Karamarda (C. carandus L.) are sour to taste, guru (heavy for digestion), ushna virya, imparts taste, increases kapha dosha and alleviates Trishna (thirst). The ripe fruits are laghu, improves taste perception, reduces vata and pitta dosha.³ The fruits have been reported to contain carissol, an epimer of α-amyrin, lupeol, oxalic, tartaric, citric, malic, malonic and glycolic acids, carissic acid, carindone, ursolic acid, carinol, ascorbic acid.⁴ Previous researches on Karamarda (C. carandus L.) have reported potential pharmacological activities like anti-cancer, anticonvulsant, anti-oxidant, analgesic, anti-inflammatory, anti-ulcer, anti-helmintic activity, cardiovascular, antinociceptive, anti-diabetic, antipyretic, hepatoprotective, neuropharmacological, and diuretic activities, antimicrobial activities and cytotoxic potentials, in-vitro anti-oxidant activity and many others.⁵

The fruits are included under Hrdya dashemani (group of ten cardioprotective herbs)⁶ and hence probably could have an effect on the heart. The cardiotonic activity and prolonged blood pressure lowering effect of the root was previously reported⁷ but no works have been carried out on its effect on coagulation. Coronary atherothrombotic diseases (CAD) include diseases of the coronary artery, peripheral vascular disease, cerebrovascular disease and heart failure. The important risk factor for CAD is formation of atherosclerosis leading to thrombosis. Oral antiplatelet drugs are used for primary and secondary prevention in many of these diseases. It is reported that patients with efficient antiplatelet therapy can reduce stroke and death rates by 25%.⁸ Hence evaluating the potential of Karamarda (Carissa carandus L.) against coagulation would contribute to its use as a dietary anticoagulant in thrombotic disease risk patients. Thus, in the present study, the anticoagulant activity of Karamarda (Carissa carandus L.) fruits extract was investigated by in-vitro method.

Materials and Methods

Collection, preparation and extraction of plant materials

The fruits of Carissa carandus L. were collected from its natural habitat, Honnavara, Karnataka, identified and a voucher specimen was deposited at the department museum for further reference. The fruits were chopped into smaller pieces, shade dried and stored in an air tight container. The fine powder of Karamarda (C. carandus L.) was prepared and stored in an airtight container till further use. The aqueous extract was prepared using maceration technique.⁹ Ten grams of the dried powder was weighed and then suspended in 200 mL of distilled water in a conical flask and allowed to stand for 24 hours with intermittent shaking. Then the mixture was filtered and evaporated to obtain the final aqueous extract. The extract was stored at 4o C and used for further studies. (Figure 1)

Stock solution

The aqueous extract was dissolved in required amount of distilled water to obtain the final concentration of 500 mg/mL and 250 mg/mL.

Collection of blood sample

The blood sample was obtained from healthy volunteers (n=10) after obtaining written informed consent. The blood sample was drawn from superior cubital vein of right arm of each person using disposable polypropylene syringe. Later the blood was dispensed into the polypropylene container containing 3.8% tri sodium citrate to prevent the process of clotting (9 parts of blood to 1 parts of tri sodium citrate). The blood sample was immediately centrifuged at 2500 rpm for 15 minutes to separate blood cell and plasma. The plasma was separated and used for the study.¹⁰

In-vitro anticoagulation activity by Plasma recalcification assay

To a clean test tube, 200 µL of plasma, 100 µL of aqueous extract of Karamarda (C. carandus L.), at different concentrations were added. 300 µL of 25 mm CaCl2 was added to the above mixture. All the test tubes were shaken, mixed well and incubated for one minute at 37°C in an incubator. For control, 200 µL of plasma, and 300 µL of 25 mm CaCl2 were taken in a test tube and incubated for a minute at 37°C in an incubator. (Table 1)

A stopwatch was used to record the time taken for clot formation. The test tube was tilted every 5 seconds at an angle of 45o to recognize the clot. The activity was expressed in terms of clotting time in minutes.¹⁰ (Figure 2). Data are expressed as mean ± standard deviation (SD).

Results

The present study was carried out to evaluate the effect of aqueous extracts of Karamarda (C. carandus L.) as an anticoagulant in blood samples of normal individuals by using in-vitro anticoagulation activity by Plasma recalcification assay. Results demonstrated that C. carandus L. extracts in different concentrations inhibit clot formation and thereby increases recalcification time (Table 2).

Discussion

Natural anticoagulant agents that inhibit coagulation process are vital for atherosclerosis prevention and in coronary artery disease.¹¹ Plasma recalcification assay, an in-vitro method is the most suitable and inexpensive screening assay to study anticoagulation.¹² Hence this method was adopted for the present preliminary study. These results conform with the observations that have been previously noticed with Fagonia arabica aqueous extracts.¹² Previous studies on various herbal extracts have shown potent anticoagulant activity.^10.11,13 In the present study, the aqueous extracts inhibited clot formation as compared to the control (2.55 ± 1.57 min). This may be attributed to the presence of phyto compounds in the extracts. Previous studies have shown that Karamarda (C. carandus L.) fruits possess phenolic compounds, like p-coumaric acid, flavonids like epicatechin, epicatechin gallate, quercetin and other compounds like l-ascorbic aicd, lupeol, carissol, ellagic acid.¹⁴ In a previous study, isohopane triterpene was found to possess antithrombotic and antiplatelet activities¹⁵ and Carandiol isolated from the leaves of Carissa carandus L, is an isohopane triterpene.¹⁶ A previous study revealed that p-coumaric acid isolated from C. carandus possesses anticoagulant activity.¹⁴ Anticoagulant, antithrombotic drugs are pivotal in the prevention and/ or treatment of thrombotic disorders. Secondary metabolites from herbs are a prospective source of anticoagulant drugs.¹³ Thus the present preliminary study revealed the anticoagulant potential of Karamarda (C. carandus L.) fruit extract.

Conclusion

Karamarda (C. carandus L.) aqueous extract (250 mg/ mL & 500 mg/mL) was evaluated for anticoagulation activity based on Recalcification time (RT) assay in an in-vitro system. From the preliminary study, it was observed that fruits of Karamarda (C. carandus L.) possess potential anticoagulation activity. Further investigations may be carried out to test the intrinsic and extrinsic coagulation cascade for better understanding of the mechanism.

Conflicts of Interests

None.