Urutan Kerja :

1. Kolagen, diambil dari limbah kan (isolasi)

2.1 Material
Collagen was extracted by the flatfish (P. olivaceus)
collected from South Korean seas. Type I collagen from
porcine skin was purchase from Matrixen TM , Republic of
Korea. High MW marker was obtained from Fermentas, (St.
Leon-Rot, Germany). SA obtained from Sigma-Aldrich, USA
was dissolved to obtain 3% (w/v) solution to be used in
scaffolds fabrication. Four molecular range of COSs were
purchased from Kittolife, Republic of Korea. EDC, Nhydroxysuccinimide (NHS), 2-morpholinoethane sulfonic
acid (MES), fluorescein diacetate (FDA), and collagenase
type I (125 CDU/mg), glutaraldehyde and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)
(MTT) were purchased from Sigma-Aldrich, USA.
Dulbeccos minimum Eagles medium (DMEM), Fetal
bovine
serum
(FBS),
trypsin
(250
U/mg),
penicillin/streptomycin and other materials used in cell
culture experiment were purchased from GIBCO TM ,
Invitrogen Corporation, USA. All other chemicals and
solvents were of analytical grade, and water used in
experiment was deionized.
2.2. Preparation of fish skin and isolation of collagen
P. olivaceus fish were collected from South Korean sea and
the skins were mechanically detached form the fish. The
skins were descaled by sharp knife and desalted by
washing with cold water at 4 C for one day and cut into
small pieces. Acid-soluble collagen (ASC) was extracted
from the prepared skin following the method described by
Singh [33] with slight modifications. All steps of the
procedure were carried out at 4 C with gentle stirring.
Non-collagenous proteins were removed with 0.1 M NaOH
at a solid to solution ratio of 1:10 (w/v) for 3 days. The
skins were then washed with dH 2 O until the washed
water become neutral or faintly basic pH. The skins were
defatted with 10% butyl alcohol with a solid to solution
ratio of 1:10 (w/v) for two days with every 12 h of

changing new solution and thoroughly washed with dH 2

O. Then skins were suspended in 0.5 M acetic acid with a
solid to solution ratio of 1:10 (w/v) for 3 days and the
suspension was centrifuged at 16,000 g for 30 min at 4
C. The supernatant was salted out by adding NaCl to
obtain final concentration of 0.9 M. The resultant
precipitate was collected by centrifugation at 16,000 g
for 1 h and then dissolved in 0.5 M acetic acid. The
solution was then dialyzed against 0.1 M acetic acid for 3
days and dH 2 O for 3 days. Dialysate was lyophilized and
was referred as ASC.
2. Bikin nanokatalis Fe3O4@kolagen
The method used for the preparation of Fe 3 O 4
nanoparticles was a modified version of a method used in
a previous procedure. [14] Typically, sodium alginate (100
mg) was dissolved in 750 mL water under vigorous stirring
for 30 min, and subsequently, 0.97 g of ferrous chloride
tetrahydrate (FeCl 2 4H 2 O) and 1.68 g of ferric chloride
hexahydrate (FeCl 3 6H 2 O) were added. A clear solution
was obtained after stirring for another 30 min. The
formation of Fe 3 O 4 was induced by the drop wise
addition of aqueous ammonia solution, which increased
the pH from 2.2 to 10. Finally, Fe 3 O 4 nanoparticles were
collected using a magnetic stone, washed with water and
methanol repeatedly to remove excess ammonia, and
stored in absolute ethanol to prevent oxidation of samples.
The method used for coating a silica layer on the Fe 3 O 4
nanoparticles was a modified version of a previously
reported method. [15]
Typically, the synthesized Fe 3 O 4 nanoparticles were
suspended in absolute ethanol (1.25 mg Fe 3 O 4 /mL
ethanol). Subsequently, 0.8 mL of 28% aqueous ammonia
solution and 2 mL of water were added to 20 mL of Fe 3 O
4 in ethanol, and this was followed by sonication for 5 min.
A magnetic stir bar was placed into the reaction vial just
before starting the slow addition of tetraethyl orthosilicate
(TEOS, Si(OC 2 H 5 ) 4 ), and the resulting Fe 3 O 4 /silica
sample was separated using magnetic force. The sample
was then washed with ethanol and water several times to

remove excess TEOS, ammonia, and non-Fe 3 O 4

containing silica. The samples were dried under vacuum
overnight.
The method used for the functionalization of the Fe 3 O 4
@silica nanoparticles with TBD was a modified method
version of a method presented in a previous paper. [16] As
shown
in
Figure
S7,
first
trimethoxysilylpropoxymethyloxirane (i.e., an epoxycontaining silane) was grafted onto the silica surface as
follows: Toluene (30 mL) was added to a pre-degassed
round bottom flask preloaded with dried Fe 3 O 4 @silica
powder (0.5 g) under N 2 , and this was followed by the
addition of trimethoxysilylpropoxymethyloxirane (2 mL).
The resulting mixture was refluxed at 1108C for 24 h, and
the product was washed with toluene several times before
being dried under vacuum. Next, 0.4 g of the dried sample
and 0.3 g of 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD)
were suspended in 15 mL of toluene, and the solution was
stirred for 8 h at room temperature. The sample was
collected and washed with toluene several times to
remove the excess TBD, and dried under vacuum
overnight.
3. Bikin
senyawa
turunan
benzimidazole
dengan
Fe3O4@kolagen
2.1. Materials and methods
All chemicals were purchased from Merck, Fluka and
Sigma-Aldrich companies and were used without further
purification. All reactions and the purity of benzimidazole
and benzothiazole derivatives were monitored by thinlayer chromatography (TLC) using aluminum plates coated
with silica gel F254 plates (Merck) using ethyl acetate , nhexane and methanol as eluents. Melting points were
determined in open capillaries using an Electro thermal
9100 instrument. IR spectra were recorded on a Shimadzu
FT-IR400s. 1H NMR spectra were recorded on a Bruker 400
Ultrasheild NMR and DMSO-d6 was used as solven
.
2.2. General procedure for the synthesis of benzimidazoles
derivatives

A mixture of aromatic aldehyde (1.0 mmol ), 1,2phenylendiamine (1.0 mmol) were employed as reactants
in the presence of catalytic amount of Fe 3 O 4 @Collagen
(50 mg) and 4 mL of EtOH as solvent was stirred
at room temperature for 1060 min, the progress of the
reaction was monitored by thin-layer chromatography
(TLC). the catalyst washed with EtOH and after drying
could be ready to use for the next reaction without loss in
activity. After evaporation of the solvent, the resulting
solid product was recrystallized from ethanol to obtain
pure product. The products were characterized by FT-IR
spectra and melting point.
2.3. General procedure for the synthesis of benzothiazole
derivatives
A mixture of aromatic aldehyde (1.0 mmol ), 2aminothiophenol (1.0 mmol) were employed as reactants
in the presence of catalytic amount of Fe 3 O 4 @Collagen
(50 mg) and 4 mL of EtOH as solvent was stirred at reflux
for 35140 min, the progress of the reaction was
monitored by thin-layer chromatography (TLC). the
catalyst washed with EtOH and after drying could be ready
to use for the next reaction without loss in
activity. After evaporation of the solvent, the resulting
solid product was recrystallized from ethanol to obtain
pure product. The products were characterized by FT-IR
spectra and melting point.