What type of anticoagulant do you mean? Is it anti-thrombin or anti-platelet?

Plant extract can be prepared and tested on blood samples and then spectroscopic determination or any other bioassay.

for anti-thrombin you can use thrombin time assay as follows:

Thrombin time assay in vitro

The antithrombin activity of LSE was ascertained using thrombin time (TT) assay in vitro. All procedures were carried-out according to the standard protocols provided with Thromboclotin® reagent and the coagulometer manual. A mixture of fresh citrated plasma and LSE samples was incubated for 3min in the coagulation analyzer wells. Then, thrombin reagent was added and the time until coagulation starts was recorded (Abdualkader et al., 2011, Schmied et al., 1995). The percentage increase in thrombin time (%TT) was calculated from the following equation:

Another test for anti-thrombin is the amidolytic assay:

Amidolytic activity: All reagents used for this experiment were

prepared in phosphate buffered saline- bovine serum albumin buffer

(PBS-BSA, pH 7.4) which contains 0.12 M NaCl, 0.01 M sodium

phosphate, 0.01% sodium azide and 0.1% bovine serum albumin.

Then, thrombin reagent and thrombin substrate S-2238 were prepared

in PBS-BSA to a final concentration of 0.6NIHU thrombin/ml and 100

μM, respectively [15].

Amidolytic assay was carried out by adding volumes of 50 μl of

thrombin reagent to equal volumes of different dilutions of LSE in the

96-well plate. The plate was shaken gently and incubated for 10min

at 25°C in the microplate reader. Thereafter, 100 μl of the substrate

was pipetted and the mixture was agitated. The absorbance at 405nm

(A405) was monitored for eight hours at 5-minute intervals [15]. Same

procedures were repeated using the PHS as a negative control. Reaction

buffer PBS-BSA was considered as a control reaction.

The percentage inhibition (% inhibition) was found out from the

following equation:

Absrobance of control % inhibition Absorbance of LSE 100

Absorbance of control

 − 

=  ×

 

The protein concentration of LSE that inhibits 50% of the enzyme

activity (IC50) was determined from the curve resulted from plotting %

inhibition against protein saliva concentrations.

Kindly find the attached

Season Variation and Starvation Period Influence on the Antithrombotic

Activity of Leech Saliva Extract From the Medicinal Malaysian Leech,

Hirudinaria Manillensis

Thank you Abdulrahman brother. Your expertise is very vast.

Dear Abdulrahman brother I am unable to download the file regarding Leech Saliva. Can you please mail me this file my email ID is [email protected]