Indo. J. Chem.

, 2014, 14 (1), 71 - 77
INHIBITION KINETICS OF Sida rhombifolia L. EXTRACT TOWARD XANTHINE OXIDASE BY
ELECTROCHEMICAL METHOD
Dyah Iswantini
1,,!
, M"a##a$ Y"%ian
1,&
, S$i M"%i'ani
1
, an( T$i)a(i%a
1,
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University,
Jl Agatis !ampus "#B Darmaga, Bogor, $est Java, %&&'(, "ndonesia
Biopharmaca )esearch Center, Bogor Agricultural University,
Jl *aman !encana No +, Bogor, $est Java, %&%,%, "ndonesia
Ar-)aniry State "slamic University, Banda Aceh, .+%%%, "ndonesia
)eceived August .,, .(%+/ Accepted January ', .(%0
ABSTRACT
Si d a rh o m 1 if o li a 2 is a traditional medicinal plant that has 1een 3no4n 4ith potential as antigout *he previous
research suggested that flavonoids crude e5tract of S rhom 1 if o lia had an inhi1itory activity to4ard 5anthine o5idase
1y 6%7 and a spectrophotometric measurement sho4ed that the type of flavonoids crude e5tract inhi1ition 4as a
competitive inhi1ition *he purpose of the research 4as to investigate the type of inhi1ition 3inetic of S )ho m 1if o li a 8s
ethanol e5tract 1y electrochemical method and to compare the measurements of linearity and sensitivity 1et4een
spectrophotometric and electrochemical methods *he results sho4ed that the yield of S ) h o m1ifo l ia 8s ethanol
e5tract 4as 9'.7 4ith the inhi1itory activity ranging from %+&07 to '.&97 :,((-.(( mg 2
-%
; and "C,( value 4as
9%%,<,60 mg 2
-%
Allopurinol as a control sho4ed the inhi1itory activity of %,.&-6(9,7 :(%(-0(( mg 2
-%
; and "C,(
value 4as .0,<..% mg 2
-%
"nhi1ition 3inetics of the ethanol e5tract caused a !M increase and unchange of =MA>
Based on the data, the type of inhi1ition 3inetics 4as a competitive inhi1ition, 4ith an inhi1itor affinity :?; value of
+%' 2inearity of 5anthine o5idase activity assay 1y electrochemical and spectrophotometric methods sho4ed the
range of ((%-%(( mM :)
.
@ (96'; and ((,-(6( mM :)
.
@ (966; respectively *he sensitivity of electrochemical
method 4as reported higher :(9, AA mM
-%
; than the spectrophotometric method :(((6 min
-%
;
Keywords: inhi1ition 3inetics/ Sida rho m 1i f olia 2/ 5anthine o5idase/ electrochemical method
ABSTRAK
Si d a rh o m1if o lia 2 merupa3an tanaman o1at yang telah di3etahui memili3i potensi se1agai antigout #enelitian
terdahulu menunBu33an 1ah4a e3stra3 flavonoid sidaguri memili3i daya inhi1isi terhadap enCim 5antina o3sidase
hingga 6%7 dan pengu3uran secara spe3trofotometri menunBu33an e3stra3 flavonoid tum1uhan ini mengi3uti
me3anisme inhi1isi 3ompetitif *uBuan penelitian ini adalah untu3 menentu3an tipe 3ineti3a inhi1isi dari e3stra3 etanol
her1a sidaguri dengan metode ele3tro3imia serta mem1anding3an linearitas dan sensitivitas pengu3uran antara
metode spe3trofotometri dan ele3tro3imia Dasil penelitian menghasil3an rendemen e3stra3 etanol se1esar 9,'.7
dengan daya inhi1isi mulai dari %+,&07 hingga '.,&97 :,,((-.(( mg 2
-%
; dan "C,( se1esar 9%,%,<,,60 mg 2
-%

Adapun allopurinol se1agai 3ontrol di3etahui memili3i daya inhi1isi se1esar %,,.&-6(,9,7 :(,%(-0,(( mg 2
-%
; dan
"C,( se1esar .,0,<.,.% mg 2
-%
!ineti3a inhi1isi dari e3stra3 etanol pada 3onsentrasi %(( ppm menye1a13an
pening3atan nilai !M :(,%'6 mM atau mening3at se1esar &',6+7; dari (,(',, mM menBadi (,.6%' mM, tanpa
mengalami peru1ahan =ma3s Berdasar3an hasil terse1ut dapat di3ata3an tipe 3ineti3a inhi1isi yang terBadi mengarah
pada tipe 3ineti3a inhi1isi 3ompetitif dengan nilai afinitas inhi1itor :?; se1esar +,%' Metode ele3tro3imia
menunBu33an linearitas pengu3uran yang le1ih 1ai3 di1anding3an metode spe3trofotometri, masing-masing pada
rentang (,(%-%,(( mM :)
.
@ (,96'; dan (,(,-(,6( mM :)
.
@ (,966; Sensitivitas metode ele3tro3imia :(,9, AA mM
-%
;
Buga le1ih 1ai3 di1anding3an metode spe3trofotometri :(,((6 min
-%
;
Kaa K!n"i: 3ineti3a inhi1isi/ Sida rhom1ifolia 2/ 5antina o3sidase/ ele3tro3imia
INTROD*CTION
Uric acid is final product of purine metabolism.
Abnormal conditions of uric acid metabolism will cause
* Corresponding author. Tel/Fax : !"#"$%#&!"'$!(/&!"'$!(
)mail address : d*ahprado+*ahoo.co.id
,*ah -swantini et al.
%
.
+
precipitation of sodium urate cr*stals in .oints,
a
condition termed gout. /out is caused b* an imbalance
between the production and excretion of uric acid
0%1.
+1
Indo. J. Chem., 2014, 14 (1), 71 - 77
important role in purine metabolism and functions to
catal*2e h*poxanthine oxidation into xanthine and,
xanthine into uric acid. The well 3nown xanthine oxidase
inhibitors 456-s7 is allopurinol, which is one option out of
the man* s*nthetic drugs used in modern medicine for
the treatment of gout 0"1. 8e9ertheless, the use of
allopurinol can cause side effects such as allergies,
fe9er, and gastrointestinal disorders. The side effects of
s*nthetic drug use such as allopurinol ha9e prompted
people to turn to traditional medicine that utili2es herbs
4medicinal herbs7 which is safer and more effecti9e.
Sida rhom1ifolia 2 is one of a traditional medicinal
plant that has been 3nown with potential as antigout.
-swantini et al. 0:1 reported that fla9onoids crude extract
of this plant has a high inhibitor* acti9it* of 56, ranging
from '& to (%; 4%<<#&<< mg =
#%
7. 8ot onl* in a single
composition, Sida rhom1ifolia 2 potential as antigout in
the combined formula has a higher inhibitor* acti9it*
compared to allopurinol, and this result has been
patented and has been granted 0'1. -n addition to its
potential as antigout, the inhibition 3inetics determination
of S )hom1ifolia8s extract as a drug candidate is also
one 9er* important thing. The determination of inhibition
3inetics t*pe which is formed can subse>uentl* explain
the inhibitor* mechanism formed and describe the
affinit* formed between 56 en2*me as a target with drug
candidate compounds, whether it is temporar*
4competiti9e inhibition and uncompetiti9e inhibition7 or
permanent 4non#competiti9e inhibition7.
Fla9onoids crude extract of S )hom1ifolia was
found out to show the competiti9e inhibition toward 56
0:1. =uteolin and >uercetin were reported to follow the
competiti9e inhibition against 56 0$1. A noncompetiti9e
inhibition of 56, among others, is indicated b* Fra5inus
angustifolia leaf extract 0!1 and *ephrosia purpurea root
extract 0(1, while the caulerpen*ne 4C?87 from the
extract of Caulerpa prolifera was reported to show the
uncompetiti9e inhibition 0&1.
@ethod that is commonl* and widel* used to
determine the t*pe of inhibition 3inetics is
spectrophotometr*. Apectrophotometric method is now
becoming obsolete, because it is less specific,
expensi9e, highl* sensiti9e to light and is affected b*
turbidit* 0B#%<1. Therefore, there should be a method
which is easier, accurate, rapid and sensiti9e in the
determination of the t*pe of inhibition 3inetics.
Campanella et al. 0%%1 reported that the electrochemical
method was more effecti9e and selecti9e and could
o9ercome the wea3nesses of the spectrophotometric
method. This method is 9er* promising because of the
relati9el* fast anal*sis time, which re>uires inexpensi9e
instrument, accurate, eas* miniaturi2ation, and simple
operation protocols 0%"#%:1. )lectrochemical method for
determination of uric acid which ha9e been de9eloped b*
Arslan 0%'1 using the immobili2ed uricase with
,*ah -swantini et al.
+
glutaraldeh*de on pol*aniline#pol*p*rrole 4pani#pp*7
composite film b* crosslin3ing procedure on the
surface of a platinum electrode. -9e3o9ic et al. 0%$1
de9eloped amperometric uric acid biosensor based
on
uricase with C"6" transducer and electrode modified
with Drussian blue.
The purpose of research was to in9estigate the
t*pe of inhibition 3inetics of S )hom1ifolia8s ethanol
extract using an electrochemical method and to
compare the measurements of the linearit* and
sensiti9it* between electrochemical and
spectrophotometric methods.
EX,ERIMENTAL SECTION
Mat-$ia%s
Sida rhom1ifolia =. Dlants were collected from its
natural habitat in Eogor, )ast Fa9a, -ndonesia.
Inst$"#-ntati.n
-nstuments used in this research were an e,AG
potensiostat 4)corder '%<7, and data processing
software of )chem 9 ".%.<. -n which an
Ag/AgCl electrode, a platinum dis3, and carbon paste
electrode were used as the reference, counter
electrode, and wor3ing electrode, respecti9el*, and
spectrophotometer.
,$./-("$-
#re$araions of "arbon $ase ele"rode
Dreparations of carbon paste electrode referred to
@irrel et al. 0%!1 with modification. A carbon paste was
made from a mixture of graphite powder and paraffin
li>uid with ratio ":% into one end of a glass tubing,
and
the surface was smoothed and cleaned with a piece of
waxed paper. Finall*, electrode surface was coated
with a dial*sis membrane, co9ered with n*lon fibre and
tied with parafilm.
%le"ro"hemi"al meas!remens
)lectrochemical measurements were carried out
using an e,AG potensiostat 4)corder '%<7, and
data
processing software of )chem 9 ".%.<. -n which an
Ag/AgCl electrode, a platinum dis3, and carbon paste
electrode were used as the reference, counter
electrode, and wor3ing electrode, respecti9el*. The
measurements were performed with an electrol*sis cell
containing %.B m= of basal solution of phosphate buffer
$< m@ with predetermined optimum pC. The test
Indo. J. Chem., 2014, 14 (1), 71 - 77
response after each addition were obser9ed and
recorded.
&he inhibiory assay a'ains ()
The inhibitor* assa* were measured at optimum
conditions ha9e been determined pre9iousl*. The potent
inhibitor* acti9it* 4;7 of the crude extract was
determined b* changes in the 56 acti9it* which is
analogous to current 4-7 before and after the addition of
extract. )xtract concentration 9aried in the range of
$#"<< mg =
#%
. -n addition, the inhibitor* acti9it* of
allopurinol as a control was also determined b* the same
procedure.
&he Kinei" inhibiion assay a'ains ()
The assa* was onl* done on the extract
concentration closest to the -C$< 9alue. The t*pe of
extract inhibition 3inetics was determined b* anal*sis of
the =inewea9er#Eur3 plot. This 3inetics stud* was carried
out in the absence and presence of extract with 9ar*ing
concentration of xanthine as the substrate.
S$e"ro$hoomeri" meas!remens
Apectrophotometric measurement referred to
Tamta et al. 0%(1 which was modified for the
determination of 9elocit* assumed as 56 acti9it*. -nto a
tube was inserted %.B m= phosphate buffer $< m@ pC
(.$. 6ne milliliter xanthine and <.% m= xanthine oxidase
<.% U/m= were added, and then the solution was
incubated at room temperature for '$ min. After the
incubation, % m= CCl <.$& @ was immediatel* added to
terminate the reaction. The absorbances of the mixed
solution were measured at "!B.$ nm.
RES*LT AND DISC*SSION
E0t$a/ti.n
The dried sample *ielded B.&%; of crude extract.
)thanol was used as extracting material because it has
two groups with different polarit*, i.e. the h*drox*l group
which is polar and the al3*l group which is non#polar.
Dolar and nonpolar compound in the sample are
expected to be extracted into the ethanol in the presence
of the two groups.
Inhi1it.$y Assay .2 th- E0t$a/t a3ainst XO
Derformance of en2*me acti9it* is generall*
affected b* temperature, pC, and substrate
concentration. Therefore, optimi2ation of 56 acti9it* was
done to stud* the effects of these parameters on 56
acti9it*. 6ptimi2ed parameters include temperature
4"$#:$ HC7, pC 0!#B1, and substrate concentrations
4<.%<#%.<< m@7. Eased on those contours 4data not
,*ah -swantini et al.
Ta1%- 1. Dotent inhibitor* effect 4;7 of se9eral
concentration of extract on the acti9it* of
xanthine
8o
0S )hom1ifolia1 Current I-pa -nhibitor* )ffect
4ppm7 4JA7 4;7
% < %.%(: <
" $ %.<%: %:.!'<
: %< <.&:: "&.B&!
' "< <.&%< :<.B'!
$ $< <.('( :!.:%(
! %<< <.$%: $!."!!
( "<< <."<: &".!B'
Ta1%- . Dotent inhibitor* effect of se9eral
concentration of allopurinol on the acti9it* of
xanthine
-nhibitor*
)ffect
4;7
<
%$."$B
"$."$B
:".$<<
'%.B&:
$&.<%(
0allopurinol1 Current I-
4ppm7 4JA7
< %.%!<
<.% <.B&:
<.$ <.&!(
% <.(&:
" <.!(:
: <.'&(
' <.::(
8o
%
"
:
'
$
!
(
pa
shown7, the optimum conditions for each of the
parameter temperature, pC and xanthine concentration
was :< HC, pC (.$, and %.<< m@. Kong et al. 0%&1 was
reported the condition of similar pC. As for the
other
different measurement conditions, it was reported
b*
CeCngi2 et al. 0&1, that is, at a temperature of :( HC
and pC B.<. The differences in the optimum conditions
for the measurement of temperature and substrate
concentration are caused b* different methods,
tools
and measurement time.
The potential of ethanol crude extract as antigout
was determined b* obser9ing the inhibitor* acti9it* of
the extract at 9arious concentrations against 56
acti9it*. Anal*ses of these 9arious concentrations were
aimed to in9estigate the effects of the increase in
the
concentration on the inhibitor* acti9it*. -n addition,
concentration 9ariation will facilitate and pro9ide
flexibilit* to the selection of the extract concentration to
be used as a candidate on 3inetics inhibition assa*.
The results of extract inhibition effect can be seen
in Table %, which shows that there is an increase in
inhibition effect in line with the increase in
extract
concentration. =inear relationship between the
inhibition effect and extract concentration is shown
b*
the decrease of current 4"7 which is analogous to 56
acti9it* after the addition of extract at 9arious
+&
Indo. J. Chem., 2014, 14 (1), 71 - 77
Fi3 1. Lelationships between xanthine concentration and current responses as 56 acti9it*
method showed an inhibitor* acti9it* of '&#(<.(%; at a
concentration of %<<#&<< mg =
#%
.
The measurable differences of the inhibition effect
in this stud* and -swantini et al. 0:1 were presumabl*
because the spectrophotometric measurement which is
strongl* influenced b* the concentration of test solution
so that the response becomes less good. -t can be
indicated that the electrochemical method can respond
to the inhibitor* acti9it* of extracts better compared to
the spectrophotometric method. According to Thuong et
al. 0%B1 a compound is said to be acti9e if it has a 9alue
of -C$< less than %<< mg =
#%
. The results showed the -C$<
9alues of extract of B%.%$M$.(' mg =
#%
.
Inhi1it.$y Assay .2 A%%.4"$in.% a3ainst XO
As comparison in this stud*, the inhibitor* acti9it*
and -C$< of allopurinol 4Table "7 were also determined.
The anal*sis showed that allopurinol had a stronger
inhibitor* acti9it* with -C$< 9alue of ".'$M"."% mg =
#%
.
6ther studies on allopurinol inhibitor* acti9it* indicated
that -C$< allopurinol which is 9aried, among others, !.%
Jg m=
#%
0"<#"%1, :.(' Jg m=
#%
0""1 and '."B Jg m=
%
0":1.
This indicated that the electrochemical method
carried out in this stud* ga9e a 9alue of -C$< which was
relati9el* small compared with the spectrophotometric
method reported b* Umamaheswari et al. 0"<1 and A2mi
et al. 0""1. Cowe9er, se9eral other studies ha9e also
reported the -C$< 9alue which was relati9el* smaller at
<.! Jg m=
#%
0"'1. -C$< 9alues in some of these studies
ma* be different because it is influenced b* differences
in obser9ing conditions 0"$1.
Lin-a$ity an( S-nsiti)ity M-as"$-#-nt
)ffect of substrate concentration on the 56 acti9it*
can be determined b* measuring the acti9it* of 56 with
9ar*ing concentrations of xanthine as the substrate. Fig.
% which is identical to the @ichaelis#@enten cur9e shows
the relationship xanthine concentration and 56 acti9it*.
,*ah -swantini et al.
+5
Leaction catal*2ed b* the en2*me was occured in
two
phases. First, if the substrate concentration is low, the
en2*me acti9e sites did not full* interact to the
substrate. Aecond, when the substrate concentration
increases, the acti9e sites is bound entirel* b*
substrate and at this time the en2*me has
been
wor3ing at full capacit* so that the addition of substrate
does not affect 56 acti9it* 0%<1. Nhen the
concentration of xanthine is less than %.<< m@,
the
reaction is in the first phase and when the
xanthine
concentration is greater than %.<< m@, 56 acti9it* is in
the second phase.
Fig. " shows the linear relationship between
substrate concentration and current responses as
56
acti9it* b* the electrochemical method in the range
of
<.<%#%.<< m@. =inear range obtained in the
electrochemical measurement is better when compared
with the results obtained with the
spectrophotometric
method. The spectrophotometric measurement was
carried out at the maximum wa9elength 4Omax7 "!B.$<
nm and showed a linear measurement in the substrate
concentration range of <.<$#<.(< m@ 4Fig. :7.
Apectrophotometric measurement ga9e high
absorbance 4P"7 at the substrate concentration P<.(<
m@ in the calibration cur9e of xanthine, that
indicates
the less accurac* and precision of measurement.
Therefore, the electrochemical method can respond
anal*te more specificall* and in the wider concentration
range than the spectrophotometric method.
Aensiti9it* is the ratio of change in response per
unit change in anal*te concentration 0"!1. Aensiti9it*
can be determined based on the slope 9alue of
the
Indo. J. Chem., 2014, 14 (1), 71 - 77
Fi3 . =inearit* between xanthine and 56 acti9it* 4Qmax7 b* electrochemical method
Fi3 &. =inearit* between xanthine and xanthine oxidase acti9it* 4Qmax7 b* spectrophotometric method
determining the 9elocit* of en2*matic reactions that form
the basis for determining the t*pe of inhibition 3inetics.
The other researches b* electrochemical methods were
reported to gi9e 9ar*ing sensiti9it* 9alues. A smaller
sensiti9it* 9alue, among others, was reported at
<.$$ JA m@
#%
0"B1 and <.<!" JA m@
#%
0:<1. Nhile the
higher of sensiti9it* 9alues was reported b* Rhao et al.
0:%1 of "B.$ JA m@
#%
. The electrochemical method can
also be reported more effecti9el* than the
spectrophotometric method in determining the t*pe of
inhibition 3inetics because electrochemical method
re>uires a shorter anal*sis time, higher sensiti9it* and
the amount of material re>uired is also relati9el* less
than the spectrophotometric method.
,*ah -swantini et al.
+6
Kin-ti/ ,$.4-$ti-s .2 XO
Concentration of selected extracts in inhibition
3inetics assa* was %<< ppm. The selection was based
on its high inhibitor* acti9it* 4P$<;7 and the
closest
concentration of -C$< extract 9alues that had been
obtained pre9iousl*. Gualitati9e anal*sis of the
en2*matic reaction is generall* carried out b*
@ichaelis#@enten cur9e, but the cur9e is 9er* difficult to
determine the 9alue of K@ and Qmax. Therefore, in this
stud* the determination of K@ dan Qmax 9alues was
carried according to =inewea9er#Eur3 plot 4Fig. '7.
,etermination of the inhibition 3inetics t*pe was
Indo. J. Chem., 2014, 14 (1), 71 - 77
Fi3 5. =inewea9er#Eur3 plot of 56 inhibition with 9arious concentrations of xanthine, addition of %<< ppm of ethanol
extract of Sida rhom1ifolia =., without ethanol extract and =inewea9er#Eur3 transformed data were plotted and
followed b* linear regression of the point
@ichaelis#@enten constant 4K@7 and maximum 9elocit*
4Qmax7 which is analogous to a maximum current 4"max7.
Eased on the anal*sis of =inewea9er#Eur3 plot 4Fig. '7, it
resulted a significant change of K@ 9alue and 9er* small
Qmax change. The 3inetic pattern that forms after the
addition of extracts as inhibitors resulted in an increase
in K@ from <.<&$$ m@ to <."(%& m@, an increase of
!&.(:;. The "max 9alue decreased from <.&"::& to
<.&":"$ and the small decrease in the "max 9alue could
be assumed there was no change 0:"1. Eased on the
results it can be concluded that ethanol crude extract of
this plant leads to the t*pe of competiti9e inhibition
3inetics as can be concluded from similar Qmax and
different Km 9alues 0::1.
)lectrochemical measurements gi9e a smaller
9alue of K@ and a higher 9alue of Q@ax which were
pre9iousl* obtained on spectrophotometric
measurements b* -swantini et al. 0:1. K@ is a measure of
substrate affinit* for the en2*me. The smaller the K@
9alue obtained on electrochemical method shows that
this method can measure the substrate affinit* of the
en2*me e9en at relati9el* lower concentrations than in
the spectrophotometric measurements.
The inhibitor affinit* 9alue 4S7 of the en2*me can be
determined b* calculating the ratio between the 9alue of
!M with inhibitors and !M 9alues without inhibitor. The
inhibitor affinit* 4S7 of extract was :.%&. These conditions
indicate the existence of strong competition between the
extract and the substrate to bind with the acti9e sites of
en2*me. The Competiti9e inhibition 3inetics of S
)hom1ifolia8s ethanol extract found in this stud*
reinforce the pre9ious research b* -swantini et al. 0:1
which explains a competiti9e inhibition of S
)hom1ifolia8s fla9onoids crude extract with smaller S
9alue of this research, that is, ".:".
(.
CONCL*SION
&.
The S )hom1ifolia8s ethanol extract has a
potential as 56 inhibitor as indicated b* its inhibitor*
,*ah -swantini et al.
acti9it* up to &".!B; 4"<< ppm7 and -C$< T%<< mg =
#%
.
-nibition 3inetics of the ethanol extract caused a change
of K@ and similiar of Qmax. Thus, the t*pe of inhibition
3inetics leads a competiti9e inhibition, with an inhibitor
affinit* 4S7 9alue of :.%&. )lectrochemical and
spectrophotometric measurements showed a linear
region of each measurement in the range of <.<%#%.<<
m@ and <.<$#<.(< m@. The sensiti9it* of
electrochemical method in 9elocit* measurement was
reported to be higher 4<.B$ JA m@
#%
7 than the
spectrophotometric method 4<.<<( min
#%
7.
)lectrochemical method pro9ides fast anal*sis time,
high sensiti9it*, eas* operation, and inexpensi9e
method in determining the t*pe of inhibition 3inetics.
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