CHAPTER 2 METHODOLOGY 2.1 Chemical Reagent D (+)





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2.1 Chemical Reagent



D (+) – Glucose
monohydrate was produced by a German company, Merck. Besides, Arginine, Bovine
serum albumin (BSA) and Bradford reagent Kit were obtained from Sigma Aldrich,
USA. In addition, Normal human plasma pool (Pool Norm), C.K. PrestÒ2 reagent
and Calcium chloride, CaCl2 0.025M were purchased from Diagnostica Stago, France.
Distilled water was sourced in-house.



2.2 Equipment and Material



Thrombin time assay was recorded using
STAGO STart 4 coagulation analyser (Diagnostica Stago S.A.S, France).
protein concentration was analysed using Spectramax M2 multi-mode microplate
reader spectrophotometer (Molecular devices, Germany). Microplate was purchased
form (Greiner Bio-One company, Austria). Universal 32R Centrifuge (Hettich
ZenTrifugen, Germany) was used for centrifugation. Thermal analysis was
performed using Differential Scanning Calorimeter (DSC) (Metler Toledo Star
System, Greinfensee, Zurich). Freeze drying was performed using shelf freeze
dryer (LyoStar 3 SP Industries Inc, Chicago, Illinois). Nalgene Rapid Flow 0.45µm 75mm bottle top filter (500mL) and
Falconä 50mL conical
centrifuge tubes (Thermo Scientific, USA) were used during filtration and
extraction process. Filtration of leech saliva extract was performed inside
Forma 1400 series biological safety cabinets (Thermo Electron Corporation, USA).
Heidolph MR 3001 magnetic stirrer (Sigma Aldrich, USA) was used to mix the
stabilizer and LSE. All instruments available at Cyberjaya University College
of Medical Sciences (CUCMS) except for freeze dryer which were lended by
Nuclear Agency of Malaysia (ANM) and coagulameter (Diagnostica Stago STart® 4
Hemostasis Analyzer, Asnieres, France) and Multi Detection Microplate Reader
(SpectraMax M5 Molecular Devices, CA, USA) which lend by Biopep Sdn. Bhd.




2.3 Site of Experimental study



Laboratory of Faculty
of Pharmacy, Cyberjaya University College of Medical Sciences (CUCMS), Malaysian
Nuclear Agency (MNA) Bangi, Biopep Malaysia Sdn Bhd operated at University
Putra Malaysia – Malaysian Technology Development Corporation (UPM-MTDC)
Technology Centre III, Serdang, Selangor.



2.4 Leech Sampling and Saliva Collection



Leech sampling, feeding, extraction,
filtration and purification were conducted by a group of people working with
Biopep Malaysia Sdn Bhd at University Putra Malaysia-Malaysian Technology
Development Corporation (UPM-MTDC).



2.4.1 Leech Sampling



Leeches were collected by local supplier from
fresh stream water in Peninsular Malaysia. The leeches were maintained in
well-aerated plastic containers filled with dechlorinated tap water and were
kept in a specific room for approximately 2 to 3 weeks before extraction
process. The temperature of the room was maintained at 25 ± 2°C.
A 10ml container was used to accommodate the leeches throughout the study
period.The dechlorinated water was regularly changed every 2-3 days (Alaamaa et al., 2011).



2.4.2 Leech Feeding



The starved leeches were fed with
phagostimulatory solution (PHS) that consist of 0.001M arginine with 0.15M
sodium chloride (NaCl2) and maintained at 37°C.
Next, the PHS solution was filled into the cellulose sac and used to feed the
hungry leeches. The leeches were brought near the sac to allow themselves to
attach to and suck the solution inside the cellulose sac. After about 25-30minutes, the leeches
become fully satiated and they detached themselves from the cellulose sac (Abdualkader
et al., 2011).



2.4.3 Extraction
of Leech Saliva Extract (LSE)



the leeches drop down spontaneously from the cellulose sac, they were kept in a
well-closed falcon tube immersed in an ice container. This step is performed to
ensure the fully satiated leeches were completely paralysed prior to the next
step. After an incubation period of 5-10 minutes, they were forced to regurgitate the solution sucked
earlier. Leeches were squeezed smoothly from the posterior end toward anterior
mouth sucker to collect the remaining solution that left inside the leech’s



The solution that have been vomited out were then collected in clean falcon tubes and segregated
according to the colour of the fluids. Only the colourless salivary fluids were
taken for this research project. Lastly, the paralysed leeches were immersed in
warm water for 15-30 minutes to regain their activity.



2.4.4 Filtration and
Purification of LSE



The colourless salivary fluid that was
collected was centrifuged at 3000 rpm at 4°C for 10 minutes. The
supernatant fluid obtained from centrifugation process was named crude leech
saliva extract (LSE), and stored at a temperature -20 ± 5°C
for the following experimental procedures.






Thermal analysis was carried out using a Differential
Scanning Calorimetry (DSC) to determine the glass transition temperature (Tg’) and collapse temperature (Tc) of the maximally freeze-concentrate LSE.
A scan speed of 2°C/min was used
for heating and cooling process runs over the temperature ranging from 25°C
to -60°C.
Figure 1.1 Shows the differential scanning calorimeter used for thermal analysis.

1.1 Differential Scanning Calorimeter (DSC) equipment used for thermal
properties determination.


2.5.1 Sample Preparation



Different concentration of LSE with glucose formulation (w/w) with the
ratio of 1:1, 1:2 and 1:3 were prepared in a small scales amount for the
determination of thermal propertied of the samples. The samples prepared were dispensed
into aluminium crucible pan for the determination of Tg’ and Tc. Then, the pan
was hermetically sealed using universal crimper press to prevent the sample
evaporate to the surrounding environment. Next, the sample and an empty crimped
reference pan were immediately placed inside the DSC oven.



A programmed warming to the ambient temperature, cooled down to -60 °C
and subsequently heated up to 25°C at a rate of 2 °C / min was applied in this
study.  The thermal events were then analysed
in the heating scan. Each sample takes approximately 80 minutes to be analysed,
and giving a total time of 240 minutes for all the 3 samples.



2.5.2    Determination of the Glass Transition
Temperature (Tg’)



STARe V9.20 Software
was used to identify the Tg’ and Tc of the prepared samples. A graph of heat
flow versus temperature was created on the STARe V9.20 program automatically
and the midpoint of the glass transition temperature, also called as a point of
inflection was plotted on the graph. Glass transition temperature (Tg’) for
each ratio were noted and used as a reference in setting the freeze drying



Freeze Drying



Solution of a homogenous mixture of
different concentration of LSE with glucose formulation
(w/w) with the ratio of 1:1 , 1:2 and 1:3 were prepared according to the previous
study conducted by Lewis et al., (2010) and Meister & Gieseler, (2009). Besides, peptides content in the fresh LSE obtained from
Biopep Sdn Bhd (62.2mg/ml)
was also used as a reference in selecting the suitable formulation
concentration (w/w) to produce a stable freeze dried products.



Stabiliser Solution Preparation



glucose (46.65 mg) which was obtained from Merck company was weighed using an
analytical balance and transferred into 250ml volumetric flask. Then, distilled
water was added into the flask until it is about ½ full, capped and inverted
several times to dissolve the glucose particles. The dissolving of glucose took
place for about less than 2 minutes. Next, distilled water was added until the
liquid approaching the etched line on the neck of the flask. Again, the flask
was capped and inverted several times to ensure thorough mixing of the sugar
solution. The final volume of the solution prepared (solution A) was 250ml with
a concentration of 186.6mg/ml.



solution for the mixture of the LSE and glucose formulation with a 1:2
concentration (w/w) was prepared using dilution method with the formula MaVa=
MbVb (Zamore
et al., 2003). From the stock solution A
prepared earlier, 66.67ml solution was transferred into a 100ml volumetric
flask and distilled water was added to the flask to make the total solution
prepared was100ml. The final volume of the solution was 100ml with a concentration
of 124.4mg/ml



an amount of 33.33ml of solution A was dispensed into a 100ml volumetric flask.
After that, distilled water was added up to the etched line on the neck of the
flask and the solution was swirled gently to ensure the solution is homogenous.
The final volume of the solution was 100ml with a concentration 62.2mg/ml.



2.6.2    Mixing and
Dispensing LSE preparation Solution into Vials



The glucose solution
prepared previously were mixed with the fresh LSE for this study purposed.
Glucose solution (60ml) from the stock solution A prepared previously was mixed
with 60ml of fresh leech saliva extract (LSE). The mixture was mixed thoroughly
using magnetic stirrer at a rate of 200rpm for 3 minutes to get a homogenous
mixture of LSE and glucose. The resulted solution was named LSE3. Then,
the solution was dispensed in separate vials using 1000mL
micropipette, where each vial contains 2ml of the solution. Total vial
containing 2ml mixture of LSE and stabiliser with a ratio of 1 to 3 was 36



The mixture of the LSE and glucose formulation with the
concentration (w/w) of 1:2 and 1:1 were prepared according to the said procedure. The mixtures were then
dispensed into different vials for the freeze drying steps with 2ml solution
each. The solutions prepared and dispensed into the sterile vials was known as
LSE2 and LSE1, respectively.



The Effect of Freeze Drying Conditions on
antithrombin activity of LSE.



A solution of homogeneous mixture of LSE and
glucose in the vials were then freeze dried at three different temperature
setting: -19°C,
and -39°C.
The temperature selected based on the thermal analysis result obtained from the
DSC analysis. The vials were arranged close to each other on the freeze dryer
tray to prevent the vials from breaking at the end of the freeze-drying cycle.
Figures 2.2 illustrate the arrangement of the vials on the freeze dryer tray. Freeze
drying was performed in shelf freeze dryer equipped with the condenser and a
chamber with cooled shelves. This step takes about 2822 minutes (47hours) to
complete all the freeze drying cycles (freezing, primary drying and secondary



2.2 The arrangement of the vials on the freeze dryer shelf during freeze drying



Effect of Storage Conditions on Antithrombin Activity of LSE



A three-month study was performed to evaluate
the long-term stability of freeze dried LSE under different storage conditions.
Glass vials containing freeze-dried LSE were stored at two different storage
conditions: 25 ± 2ºC and 4 ± 2ºC. Percentage increase in the thrombin time (%TT)
for each sample were monitored for a period of three months to study the
antithrombin activity and stability of the freeze dried samples. Samples were
analysed at three different time points: on month 1, month 2 and month 3 (t1,
t2 and t3, respectively). All samples were kept inside a closed container to
protect from direct light during the study was performed.





2.8 Protein Quantification



quantification was carried out using UV absorption and standard protocol of Bradford
reagent method. The UV method was performed to estimate protein concentration
at A595 maximum absorption. Bovine Serum Albumin (BSA) was used as a
standard protein and distilled water was used as a blank instead of 0.15M
sodium chloride (NaCl2).



2.8.1 Standard Solution and Sample Preparation



solution of a BSA with a concentration of 5mg/ml was prepared by diluting 25mg
of BSA powder with 5ml distilled water. Next, a series of 5ml standard
solutions of BSA 20 µg/ml, 40 µg/ml,
60 µg/ml, 80 µg/ml, and 100 µg/ml each were prepared using dilution method from
the BSA stock solution. Distilled water was used as a blank. The prepared standard
solutions with different concentrations were kept and used throughout this
experiment for total protein estimation.



Freeze dried samples that were stored at two
different temperature setting for a specified period were diluted with 1ml
distilled water using 1000mL
micropipette prior to protein quantification study. The standard solutions and
Bradford reagent was taken out from the refrigerator 30 minutes before running
the assay to equilibrate to room temperature.


solution was
pipetted into first three
microplate wells,(A1, B1 and C1) by using 100µL micropipette with the volume of
30 µl each, After that, 30µL
the previously prepared standard solution with a concentration of 20 µg/ml was carefully added into the next wells
of the plate in triplicate starting from position A2,
B2 and C2, followed by the different concentration of the standard solution
(40 µg/ml, 60 µg/ml, 80 µg/ml, and 100 µg/ml) in the next wells,
from the left to the right.



After all the
wells of the micro-plate were filled with the standard solution, thirty
of the previously diluted freeze-dried LSE samples (LSE1, LSE2 and
LSE3) were then pipetted into each well in triplicate starting from
the least concentrated to the most concentrated one. Next, 200µl of bradford
reagent was carefully added to each well using a multichannel micropipette 250
µl (Figure 2.2). The mixture was incubated at room temperature (RT) for 30 minutes
prior to be inserted into the spectramax spectrophotometer.


Figure 2.3 Microplate that is pipetted with the standard
solution, samples and bradford reagent ready to be analysed.



2.8.2 Protein Reading



Spectramax M2 Pro software was used to analysed the data. After
5 minutes incubation, the micoplate was inserted into the drawer of Spectramax M2
multi detection microplate reader
and their absorbance wavelength was set at a 595nm. The absorbance of
the standards, fresh leech saliva extract and freeze-dried leech saliva extract
were measured.



that was obtained earlier was exported to Microsoft excel and a standard curve
was generated by plotting the absorbance values on the y-axis versus
concentration (?g/ml) on the x-axis. The unknown protein concentrations in the
freeze dried LSE samples were determined using the standard curve.



 Thrombin Time Assay In-Vitro



bioactivity and stability of freeze dried LSE were examined using percentage
increase in percentage thrombin time (%TT) and percentage total loss of
activity as compared to the fresh non-lyophilized LSE. Thrombin time assay was
carried out according to the standard protocols of normal plasma together with C.K.
PrestÒ2 reagent and STAGO STart
4 coagulation analyser protocol.



2.9.1 Normal Plasma Preparation



Normal human plasma pool
(Pool Norm) was reconstituted with 1.0 ml of distilled water. The prepared plasma
was incubated for 30 minutes at 18-25 ºC prior to use. According to the Pool
Norm manual, the reconstituted plasma is stable for 4 weeks at -20° C.



2.9.2     Plasma Reagent (C.K. PrestÒ 2) Preparation



One vial of reagent 2
(R2) was transferred into a vial of reagent 1 (R1), shaken gently and let to stand for 30 minutes at room
temperature (18 – 25°C). According to the C.K PRESTÒ 2 manual, the reconstituted C.K PrestÒ 2 is stable for two days at 20 ±5 °C and seven days at 2 – 8 °C.



Sample (Mixture of LSE and Normal Plasma) Preparation



The reconstituted
freeze dried samples used previously were mixed with normal plasma with a
volume ratio of 1: 4. Thirty microliter LSE was added into 120µl normal plasma
and mixed thoroughly by using mini vortex mixer for 5-10 seconds to ensure
homogenous solution is formed.



2.9.4 Thrombin Time



The mixture of plasma and LSE prepared earlier
was pipetted into a pre-warmed coagulation tubes with the volume of 50 µl each
tube. Then, 50 µl of the reconstituted C.K PrestÒ2 reagent was added and the mixture was
incubated at 37°C for 170s second. Next, a drop of 0.025M CaCl2 was
added to induce clot formation and the time until the coagulation starts was
measured by coagulometer. The normal plasma with a usual coagulation time
30-40seconds was used as a negative control. All samples were prepared in



2.9.5 Percentage Increase in the Thrombin Time (%TT)



The percentage increase in thrombin time
(%TT) was calculated using the following equation:





Samples were analysed at three different time points, on month 1, month 2 and month 3 (t1, t2 and t3, respectively). The bioactivity of the freeze dried LSE was estimated by plotting the % increase in the thrombin time (%TT) against different time point (t1, t2, t3). Fresh LSE was used as a positive control in this study.  













2.10 Statistical Analysis



Data for this study will be analysed by using
Statistical Package for the Social Science (SPSS) version 22 for Windows and
was tested with appropriate one way ANOVA and descriptive statistics. Chart and
graph were produced by using Microsoft Excel.



The effect of concentration of the glucose on the
antithrombin acitivity of the freeze dried LSE samples were tested using one
way ANOVA. The same test was used to study the effect of freeze drying
temperature setting on antithrombin activity of the sample.



Descriptive statistic such as percentage, mean and standard
error mean (SEM) were used to present the long-term storage effect on the
percentage thrombin time (%TT) of LSE. Independent T-test was used to see the
effect of storage temperature (room temperature, fridge temperature) on
antithrombin activity of freeze dried LSE.