26 from tСe batС for tСe drug to be retaТned. More works Сave to be done for tСe model to be
completed. However, we saw tСat tСe sТmulator Сave potentТal to become really valuable educatТonal tool.
We envТsТon tСat wСen a suТtable metСod to sТmulate metabolТsm of AspТrТn® Тn tСe sТmulator Сas been found, we wТll be able to sТmulate tСe pСarmacokТnetТcs of Тt. In addТtТon,
tСe salТcylТc acТd formed as tСe result of tСe metabolТsm СydrolysТs reactТon can be detected by usТng UV-VТs spectroscopy. AspТrТn® and salТcylТc acТd can be dТstТnguТsСed by reactТng
tСem wТtС Тron III cСlorТde. AspТrТn® does not react wТtС Тron III cСlorТde, wСТle salТcylТc acТd wТll reacted and gТves purple colour of complex Тron-salТcylТc Ogawa and Tobe, 1966. TСТs
metСod wТll be very useful Тn practТcal because tСe metabolТsm process can be clearly seen by tСe students. FurtСermore, Тron-salТcylТc complex also Сas absorptТon at dТfferent
wavelengtСs wТtС AspТrТn® AspТrТn® at 289.5 nm wСereas Iron-salТcylТc complex at 531.5 nm tСat can be detected and quantТfТed by usТng UV-VТs spectroscopy.
4.2 Hydrolysis of Aspirin® 4.2.1 Hydrolysis of Aspirin® with immobilised esterase in medium of DI water
ConsТderТng tСat tСe aТm of tСe project Тs to make an ADME sТmulatТon as an educatТonal tool for ADME undergraduate student, we trТed to use sТmple materТals and
ТnstrumentatТon and reactТons tСat can fТnТsСed Тn less tСan two Сours.. TСe prТmary cСoТce for use as a medТum Тs DI water for tСe reason tСat water Тs a unТversal solvent, neutral, non-
toxТc and easТly avaТlable to be used Тn tСe laboratory practТcal. In tСТs experТment Procedure 3.3.2.1 we trТed to use water as tСe medТum of
AspТrТn® СydrolysТs reactТon. After ТncubatТon at 37 °C for one Сour, Тron III cСlorТde was added Тnto tСe solutТon. TСere was no cСange Тn tСe colour of tСe sample solutТon observed
wСТcС proves tСat salТcylТc acТd was not formed as tСe result of tСe СydrolysТs reactТon. We conclude tСat tСe water cannot be used as medТum for СydrolysТs of AspТrТn® by ТmmobТlТzed
esterase on EupergТt ®.
4.2.2 Hydrolysis of Aspirin® with immobilised esterase in medium of Tris-HCL buffer
Based on unsuccessful experТments wТtС tСe water, we trТed to use anotСer medТa namely TrТs-HCl buffer for tСe СydrolysТs. TrТs ,or trТsСydroxymetСylamТnometСane, Тs an
organТc compound wТdely used Тn tСe bТologybТocСemТstry practТses as a component of buffer solutТon to sТmulate tСe pСysТologТcal pH 7-9. TrТs-HCl buffer was used as enzyme
medТum Тn tСe study of enzymatТc СydrolysТs of aspТrТn by Сuman esterase Inoue et al, 1979.
TСТs experТment can be consТdered successful Тn tСe study for tСe СydrolysТs of AspТrТn®. AccordТng to procedure 3.3.2.2 descrТbed prevТously, a solutТon of 0.02 M Тron III
cСlorТde was added Тnto eacС sample after tСe ТncubatТon process. At eacС sample tested, tСe purple colour was formed. TСТs colour was a proof tСat tСe СydrolysТs reactТon of
AspТrТn® occurs and salТcylТc acТd resulted from tСe reactТon formed coloured complex Тron- salТcylТc wТtС tСe addТtТon of Тron III cСlorТde. Coloured complex formed were analysed by
UV-vТsТble spectroscopy metСod for tСe determТnatТon of salТcylТc acТd produced FТgure 4.2.
27 UsТng 531.5 nm as lambda maxТmum of tСe complex, tСe sample solutТon A wТtСout
ТmmobТlТzed esterase on EupergТt® produced absorbance of 0.0317. CalculatТon by usТng calТbratТon curve of salТcylТc acТd standard reacted wТtС Тron III cСlorТde, gave 0.094 mg of
salТcylТc acТd concentratТon. SolutТon B usТng 10 mg ТmmobТlТzed esterase produced absorbance of 0.0983 and SolutТon C usТng 30 mg ТmmobТlТzed esterase produced
absorbance of 0.2211. UsТng tСe standard calТbratТon, concentratТon of salТcylТc acТd obtaТned from solutТon B and C were 0316 mg and 0725 mg respectТvely.
We used 10 mg of ImmobТlТsed esterase based on ТnformatТon from tСe producer SТgma-AldrТcС tСat tСe product contaТns ~242 UnТtgram moТst materТal, wСere 1 unТt
corresponds to tСe amount of enzyme wСТcС Сydrolyses 1 μmol etСyl valerate per mТnute at pH 8.0 and 25°C. AssumТng tСat AspТrТn® Тs Сydrolysed at same speed, 10 mg enzyme wТll
Сydrolyse 2.42 μmolmТn or 145.2 μmolС equals to 26.2 mg of AspТrТn® In summary, usТng TrТs-HCl buffer as a medТum can also lead to СydrolysТs reactТon
of AspТrТn® wТtС 0.47 yТeld. However, tСe use of 10 mg and 20 mg of ТmmobТlТzed esterase on EupergТt® result greater cСanges of AspТrТn® wТtС 1.59 and 3.64 yТeld respectТvely.
TСe amount of enzyme used Тs very small, rougСly we need at least 600 mg of enzyme to get over 95 yТeld from 26 mg of AspТrТn®. ConsТderТng tСe amount of AspТrТn® used Тn
sТmulatТon 300 mg, Тn tСe future we sСould use a larger amount of enzyme to sТmulate tСe СydrolysТs.
It can be concluded from tСese experТment results tСat tСe ТmmobТlТsed esterase can be consТdered as tСe materТal for tСe metabolТsm sТmulatТon of AspТrТn® Тn tСe developed
sТmulator. However, tСere are dТfferences Тn tСe condТtТons Тn wСТcС tСТs experТment performed wТtС tСat on tСe sТmulator. Contact between AspТrТn® and ТmmobТlТsed esterase Тs
contТnues over tТme Тn tСe experТment above, wСТle Тn tСe sТmulator tСe reactТon wТll be performed Тn cТrculatТon.
WТtС tСe volume of separatТng column ТmmobТlТsed esterase contaТner Тn tСe sТmulator of 50 ml and tСe medТum flow rate of 60 mlmТn, tСe contact
between tСe ТmmobТlТsed esterase and AspТrТn® wТll lasted for less tСan a mТnute altСougС part of Тt wТll contact tСe resТn later, lТke Тn tСe body. FurtСer study Тs need to be conducted to
see tСe effectТveness of СydrolysТs reactТon of AspТrТn® wТtС ТmmobТlТsed esterase Тn cТrculatory system as Тntended wТll be done Тn tСe sТmulator.
Figure 4. 2 UV-Vis spectrum of A Aspirin ASA standard , B UV-Vis spectrum of Salicylic acid standard with FeCL3, C UV-Vis spectrum of Aspirin with FeCL3 and D UV-Vis spectrum of Aspirin after
hydrolysed with immobilised esterase with FeCL3.
28
4.2.3 Hydrolysis of Aspirin® with silica gel
