Invitro Anticoagulant Activity of Nux Vomica, Sapota and Soapnut Seed Extracts

Coagulation, commonly referred as blood clotting, is a physiological  process in which blood changes from a fluid into semisolid  gel, leading to the  formation of  a blood clot.This process helps achieve   hemostasis, preventing excessive blood loss from an injured blood vessel and supporting tissue repair. Blood coagulation involves platelet activation, adhesion and aggregation followed by the formation and stabilization of fibrin network

The coagulation cascade is initiated through two primary pathways ,the intrinsic and extrinsic pathways . The extrinsic pathway is triggered  when  circulating blood comes into contact with tissue factor released  from damaged the extravascular cells, in contrast , the intrinsic pathway is activated  by factors present within  the injured blood vessels. Platelet activation occurs in response to various  stimuli, including collagen and arachidonic acid , which promote the synthesis of  Thromboxane B2 via the Cyclooxygenase 1  and Thromboxane A2 synthase enzymatic pathway (TXAS). TXB2 plays a significant role in enhancing  platelet aggregation, and facilitating platelet to platelet interactions there by contributing to thrombus formation and  increasing the risk of  thrombotic disorders.

Numerous medicinal plants have been reported to exhibit anticoagulant properties by reducing platelet aggregation, extending blood clotting time ,and influencing various coagulation pathways.Among these soapnut seeds ,sapota seeds and nux vomica seeds have shown notable anticoagulant effects. Nux vomica seeds are rich in alkaloids and chlorogenic acid ,soap nut seeds were rich in saponins, ,flavonoids and phenols where as sapota seeds were rich in flavonoids ,tannins and phenolic compounds which may inhibit clotting enzymes and increase coagulation parameters such as Prothrombin time and activated partial prothromboplastin time.

MATERIALS AND METHODS:

Plant collection and extract preparation:

Three  plant species of  Strychnos nux vomica (Nux vomica seeds) ,Sapindus mukkorossi (soap nut seeds) and  Manilkar zapota(sapota seeds) were  purchased from local Sangareddy market. 

Extract preparation of Strychnos Nux vomica seed:

Nux vomica seeds were washed cleanly , dried and crushed in a homogenizer  and sieved and further stored in a dry place at room temperature until the day of extraction. Seed powder of about 50gm was taken and  extracted with  ethanol(50%)o by using soxhlet extractor .The extract thus obtained is filtered by using whatmann filter paper and filtrate is evaporated to dryness.It is further diluted and various concentartions were prepared and used for anticoagulant studies.

Extract preparation of Sapindus mukkorossi (Soap nut seeds):

Soap nut seeds were collected  and washed cleanly , dried and outer shell is removed and kernels were collected and crushed in a homogenizer  and sieved and further stored in a dry place at room temperature until the day of extraction. Seed powder of about 50gm was taken and  extracted with  ethanol(50%)o by using soxhlet extractor .The extract thus obtained is filtered by using whatmann filter paper and filtrate is evaporated to dryness.It is further diluted and various concentartions were prepared and used for anticoagulant studies.

Extract preparation of Manilkari zapota(S

All the seed extracts were subjected to phytochemical tests to determine the presence of Alkaloids, Flavonoids,Saponins and Coumarins.

Blood collection and  platelet plasma preparation:

 A healthy volunteer of about 18-35 years old were selected and blood samples were withdrawn from the selected donor via vein puncture and collected blood is transferred into separate tubes containing EDTA to prevent clotting.Centrifuge the blood samples for 15 minutes at a rate of 3000 rpm to separate the blood cells from plasma in order to obtain pure platelet plasma (ppp) for performing Prothrombin time test.

Prothrombin Time Test:

For the plasma recalcification assay,0.2ml of plasma was mixed with 0.1ml of ethanoic plant  seed extracts at various concentrations along with different volumes of 25mm calcium chloride (Cacl₂) in a clean test tube. The mixture was incubated in a water bath maintained at 37⁰C. in the control group, the extract was substituted with an equal volume of 0.9% normal saline . Clot formation was monitored by gently tilting the test tube every 5 seconds and the time required for clot formation was measured using a stop watch. The recorded clotting time was considered as prothrombine time .

RESULTS

This study was conducted to investigate the anticoagulant potential of Nux vomica seeds, Soap nut seeds and Sapota seeds using blood samples collected from healthy individuals . The anticoagulant  activity of these plant extracts was assessed by determining their influence on  blood coagulation time

Phytochemical screening:

Table no:1

+ Presence ,  - Absence

From the phytochemical screening we found that Nuxvomica seed extracts have shown  the presence of Alkaloids,Coumarins,Flavonoids and Saponins.Sopa nut seeds have showed the presence of alkaloids, coumarins and saponins.Sapota seeds have revealed the presence of coumarins and saponins.

Anticoagulant activity

 Ethanoilc  extracts of soapnut seeds have exhibited coagulation time of about 7min and Nux vomica seed extract coagulation time was about 4min 30sec and Sapota seed extract coagulation time was 5min 24sec.    Ethanolic extract of Nux vomica seed have exhibited more clotting time when compared to soapnut and sapota seed extracts. Its activity may be due to the presence of Phytoconstituents like Alkaloids, saponins, flavonoids and coumarins  in the sample .

Anticoagulant Assay (Prothrombin Time Assay)

Table no:2