The Chelating Effect of Mungiferu foetida Water extract on Serum

The ChelatingEffect of Mungiferufoetida
Waterextracton SerumThalassemic-Patients
Ernie H. Purwaningsih* Endang Ilanani** PustikaAmalia***
Desak Gede Budi Krisnamurti*
*Department of Medical Pharmacy, Faculty of Medicine
Universitas Indonesia, Jakarta
**Department of Pharmacy, Faculty of Mathematics
and Natural Sciences (Jniversitas Indonesia
*** Department of Child Health, Faculty of Medicine (Jniversitas
Indonesia/
Cipto Mangunkusumo Hospital, Jakarta
Abstrsct: Thalassemia is still a major problem in Indonesia becquse of its high morbidity and
mortality rate. The well-lcnown chelating agent, deferoxamine, which is regularly administered,
is expensive and has many side fficts. Mangiferin, extractedfrom the bark of Mangifera indics
Linn. has beenproven in vitro, as a chelating agent to iron. The aim of this study was to prove
that the water extract of Mangifera foetida Lour leaves has o chelating ffict to serum ferritin
from thalassemic patients. This study was conducted at the Department of Medicat Pharmacy
FMUI, Department of Child Health FMUI, and Department of Pharmacy, FMIPA rJlfrom October
2009 to June 2010. Forty two samplesof serumfrom 7patients thqt have beendiluted to 200 pM,
were divided into 6 groups as follows: negative control group) the treqted group of extract,
0.375; 0.75; 1.125 mg, respectively; mangiferin 100 pg; deferoxamine 100 ps. The chelating
activity was measured by substraction betvveenthe absorbance values of serum containing this
treatment and each treqtment only, divided by serum absorbance, using spectrophotometer qt ),
280 nm. One way Anova was usedfor statistical analysis. The water extract of Mangiferafoetidt
lewes at the dose 1.125 mg showed chelating ffict toferritin serum that has no significant
dffirent to mangiferin 100 ug (p:0.735) This result showed dffirently compared to in vitro
study because in serum, ferritin contains apo-protein that binds iron tightly, thus another specific method should be createdfor detecting their binding. J Indon Med Assoc. 2011;61:321-5.
Kqtwords: Mangifera foetida, mangiferin, thalqsemia, chelating agent
J Indon Med AssocoVolum: 61, Nomor: 8, Agustus 2011
321
TheChelatingEffect of Mangifera foetida Waterextract on SerumThalassemicPatients
Efek Kelasi Ekstrak Air Mangiferafoetida.pada Serum
Penderita Talasemia
*
ErnieH. Purwaningsih*EndangHanani** PustikaAmalia'r'r'
DesakGedeBudi Krisnamurti*
rDepartemen Farmasi Kedokteran, Fakultas Kedokteran Universitas Indonesia, Jakarta
**Depdrtemen Farmasi Fakultas Matematika Ilmu Pasti Alam Universitas Indonesia,
***Departemen'^;:;:f
;'#,!,i!;iilll'f i,,,f
!i#'m,Y:;*u^Indonesia/
Abstrak: Di Indonesia, penyakit talasemia masih menjadi masalah karena tingginya angka
kesakitan dan kematian. Desferoksamin merupakan satu-satunya terapi kelator zat besi, namun
harganya sangat mahal dan efek sampingnya dapat memperburuk kondisi pasien. Alternatif
yang digunakan adalah mangiferin, hasil ekstraksi batang Mangifera indica Linn. yang terbuhi
sebagai kelator besi in vitro. Tujuan dari penelitian ini adalah membuldiknn bahwa elcstrak air
daun Mangiferu foetida Lour. memiliki efek kelator pada feritin serum penderita talasemia
secarauvixo. Penelitian dilakukanselama I0 bulan, dari Ohober 2009-Juni 2010, di Departemen
Farmasi Kedoheran, Departemen llmu Kesehatai Anak FKUI, dan Departemen Farmasi FMIPA
UI. Sebanyak 42 sampel serum dari 7penderita talasemia dengan kadarferitin yang diencerkan
menjadi 200 uM, dibagi mmjadi 6 kelompok: kontrol negatif, 3 kelompok el<strak 0,375; 0,75
dan 1,125 mg; mangiferin 100 ug; desferoksamin l00ug. Ahivitas kelasi diukur denganmembagi
hasil pengurangan absorbansi serum pada masing-masing perlakuan dengan absorbansi serum
pada spehrofotometer, lambda 280 nm. Uji statistik yang digunakan adalah One-way Anova
(p:0,05) Hasil uji menunjukkan bahwa ekstrak dosis 1,125 mg memiliki efek kelasi yang tidak
berbeda bermakna dibandingl<an mangiferin (p:0,735). Hasil tersebut berbeda dengan uji in
vitro karena besi dalam serum terikat dengan apoprotein. Pembuktian bahwa ekstrak atau
mangiftrin berikatan dengan feritin memerlukan metode pengukuran yang spestfik. J Indon
Med Assoc. 2011;61 :321-5.
Kata kunci: Mangifera foetida, mangiferin, talasemia, kelator besi
Introduction
One of severalgeneticdiseases
with increasingtendency is Thalassemia.According to data from Cipto
Mangunkusumo
Hospitalin 2008,l4l2 newpatientswere
treatedandthemortalityratewas0.8o/o.Intheyear
of 2006,
therewere3053thalassemic
patientsandthe numberhave
increased
to 5000patientsin 2008.r The surveyalsonoted
thattheprevalenceof thalassemic
carrierin Indonesiais 38%; includingdatafrom Makasar(8%), Palembang(7Yo),
andJava(3.4%).Therearetwo typesof thalasemia,
minor
type (cr-Thalasemia)
danmajortype (p-Thalassemia).
The
highermorbidity andmortality werefoundin p-thalassemia
dueto severeiron overloadbeforeand/orafter transfusion.
Deferoxamineis a chelatingagent,usuallyusedfor decreasingiron accumulationin the organs.2,3
Unfortunately,
deferoxamineis very expensive.Furthermore,the longer
durationor more invasivemethodof administrationcause
severalsideeffectssuchasarthralgia,fever,headache,myalgia,nausea,vomiting,abdominalpain,or asthma,beside
local injection site reactions.Thoseare also the reasons
why patients'complianceare low and subsequently
stop
their therapy.a
322
Therewere other attemptsto overcomethe patients
compliance.A researchon mangiferinhad beendone in
Cuba,5,6
where mangiferinwere extractedfrom the bark of
Mangtfera indica Liwt, and proven as chelating agent to
iron in vitro. In Indonesia,researchers
from Instituteof Technology Bandung(ITB)? found that the extractof Mangifera
indica (bacang)leavescontainshigherlevel of mangiferin
than othervarietiesof mango,which was 2.56%.Mangga
(mango)bacang(MangiferafoetidaLour)is easilyfoundin
WestJawa. It has specificfragrance,but usuallynot consumedin its raw form becauseof its fiber andtaste.
This study was proposedto revealthe effect of water
extract of Mangiferafoetida Lour's leavesas chelatoron
ferritin in the serumofthalassemicpatientsfrom Department
of Child HealthFacultyof MedicineUniversitasIndonesia.
In thelongrun,shouldtheeffrcacyestablished,
it is expected
that the outcomeof this studywill be able to increasepatients' compliance,and eventuallyincreasingthe survival
rate. Furthermore,
the studymay alsoenrichtheuseof evidence-based
Indonesiannaturalherbalmedicinewith standardizedindustrialization.
J Indon Med Assoc,Volum: 61, Nomor:. 8, Agustus 2011
The ChelatingEffect of Mangifera foetida Waterextracton SerumThalassemicPatients
Methods
Result
This was a cross-sectional
studyheld at the DepartmentofMedicalPharmacy
FMUI, Department
ofChild Health
FMUI, Departmentof Pharmacy,Facultyof Mathematics
andNaturalSciencesUI Depokduring l0-monthperiod,
fromOctober2009to June2010.
Manggabacang'sleaveswereharvested
from Depok,
WestJavaandhasalreadydeterminedasMangiferafoetida
Lourby LIPI Bogor.Mangiferinwaspurchased
fromChina,
deferoxamine
fromAventis@,
andstandardized
chemicalsubstancesfrom Merck@.
All serumthalassemic
patientswere
treatedat Department
of ChildHealthFMUI.
Phytochemistrycomparisonbetweenwaterextractand
ethanolextractof Mangiferafoetidacanbe seenin Tablel.
Table 1. The Phytochemistry Comparison Between the Water Extract and the Ethanol Extract of MangiferafoAida
Phytochemistry
Steroid and Triterpenoid
Alkaloid
Phenol
Flavonoid
Saponin
Water extr.
Ethanol extr.
+
;
+
+
+
+
+
Extraction Process
Four-hundred
gramsofdried mangobacangleaveswere
extractedwith waterusinginfusionmethod.The supematant wereevaporatedby rotary-evaporatorabovethewater_
bathof 60"C.Thedriedextractwereweighedanddividedby
400 gramof their simplisiato determinetheir final extract
product(the dried extractwhich yieldedby the extraction
process).Partof it wasusedfor phytochemistry
examination.Thewaterextractionwasdoneat Department
of Medical PharmacyFMIII andthe ethanolextractionat Departmentof PharmacyFacultyof Mathematics
andNaturalSciencesUI.
SpecimenCollection and Processing
The subjectswereaskedto sign an informedconsent
afterreceivingexplanation
regardingthe study.Theywere
askedto give6 mL oftheirblood. Serumfrom 7 thalassemic
patientswastakenat randomfrom24 patients.Therewere
42 samplesfor 6 treatmentgroupsasfollows:negativecontrol group,the treatmentof the extractat the dose0.375;
0.75; I.125 mg, respectively;
mangiferin100 pg, and
deferoxamine
100pg. Theextractdoseweredetermined
by
theextrapolating
ofthe doseof mangiferinl00pginvitro,5,6
otherwisetheconcentrationof mangiferinin this extractwas
assumedas2.56oh,basedon the researchby Sudiroet al.j
Seraweredilutedto achievethe concentration
ofiron 200
pM, basedon the ferritin measurement
usinglatexagglutination.8The doseof deferoxamine
weredeterminedby ln
vitro sbtdy.e
Serumof ferritin200pM werefilled in to every
cuvette,thenaddedwith treatmentasfollows:aquabidest
I
mL asnegativecontrolgroup;I 00 pL extract 0.375 mg; I 00
pL extract0.75
mg;100trrL
extactl.l25mg;100pLmangrferin
100pg; 100pL deferoxamine
100pg, andthenadded,each
sample,with aquabidest1 mL. Stinedbeforemeasured
on
spectrophotometer,
l" 280nm. No specificreagents,
except
citric acid, were usedin this study,becauseof the same
resultsasin watertreatment.The chelatingactivitieswere
measuredby meansof the differencesbetweenthe absorbanceof theserumwith thetreatmentandtreatmentwithout
serumand dividedby the absorbance
of serum.Oneway
Anovaat p : 0.05wereusedto analyzethe differences.
J Indon Med ASsoc,Volum: 61, Nomor: 8, Agustus 2011
Therangeof ferritinserumofthalasemic
patients(4 - 25
yearsold)whichin thisstudywere612.2-20.297
ng/ml measuredby a specialkit, latexagglutination,
at Clinicalpathology LaboratoryCipto Mangunkusumo
Hospital.8
Basedon deferoxamine
problemto treatan iron overloadonthalasemicpatients,thewaterextractofmangobacang
that will be usedasa complementaryand./oralternativedrug
showedtheir efficacy as describebelow. The absorbance
valuesfromthethreedoseof theextract0.37S;0.75; | .125mg
from seventimesexperimentshowedhaslinier crnves.This
resultprovedthat the experimenthasalreadybeendoneaccurately(Kolmogorov-Smirnov,
p:0.052) asshownon Figure2.
1,00
6,00
5,00
4,00
3,00
2,00
1,00
0,00
Figure 2. The AbsorbanceValues from the Three Dosesof Extract
The efficacyofthis extractwas not found at the dose
0.375mg (absorbance
value:28.44),but was found at the
dose0.75mg (absorbance20.6l)andl.l25 mg (absorbance
l7 .72).Atthedoseof 0.75mg andparticularly1.125mg,the
effrcacyshowedvery different significantly compareto the
controlgroup(p:0.022) andbut showedno significantdifferentto mangiferin100ug (absorbance
7.22)withp:0.735
(SeeFigure3).
323
TheChelatingEffect of Mangifera foetida Waterextract on SerumThalassemicPatients
densitiesweredifferent(datanot shown).Thatwastheparticularreasonwhy thechelatingactivitiesweremeasured
by
meansof their differencesas describeabove.However,in
r35
r30
at l.
orderto provethat the chelatingeffectwasproceeded
9zs
280 nm, nuclearmagneticresonqnce-mass
spectrophotom8zo
eter Q'{MR-MS)
is suggested
to be usedin the future.t0
Fts
€ro
The samereasonwas alsothe samearguedas mangiB5
ferin.Themangiferinabsorbance
couldnot be detectedat l,
320nn andtheir chelatingeflectcouldnot alsobe detected
at
l" 380 nm. This resultwas differentcompareto in vitro
6souJ".
*rton
J'e
.6r"d
Theyusedfreeiron assamples,
but we usedferritin
study.5'6
,S'
,p.t $,.o"
"+"
serumthat containsapofenitin which hasboundiron tightly.
Typesof treatment
Thebindingof mangiferinandferritin shouldalsobe elucidatedusingthe equipmentabove.
Figure 3. The Chelating Effect of Water Extract of Mangiftra
rnd
Mangiferin
100
ug
Ferritin
on
Serum
Thewaterextractof mangobacangleaveswaschosen
foetida
Compared to Control Group
in this studyto replacethe bark of Mangifera indica to prevent damagingeffectto thetree'secosystem.Furthermore,
Discussion
the leaves,particularlythe oldestleaves,arenot only easy
to harvestbut alsoeasyandcheaperto extract.Theyshowed
On stepI, theyield ofthe waterextractasa final extract
the optimalchelatingeffecton fenitin serumof thalasemic
productor the yield extractionfrom 400 gramsimplisiaof
o/o,but
patientsat the dose1.125mg.Theireffecthassignificantly
Mangiferafoetidaleaves
10
was
theethanolwas8.6
oh.T\e ethanolextractionwas also doneto prove that the
differenceto the controlgroup and showedno significant
differentto mangiferin100 pg. It was assumedthis dose
waterextracthasalsohadthe samefinal extractproductand
wouldcontainmangiferin28.8pg, accordingto Sudiroet af
phytochemistrylevel as the ethanolextract(seeTable l).
thatthecontainof mangifeinfrom Mangiferaindica.leaves
Thestudiesfrom Cubas,6and
ITBTalsousedethanolfor their
dose(100
was2.96%. Thisdosewaslowerthanmangiferin
experiments.Unfortunately,they did not publishtheir final
mg), so the effectmay be causedby the synergyeffectof
exfractproduct.Thewaterextractwerechosenon this study
severalcomponent
ofthis extractsuchassteroid/triterpenoid,
becauseofthe fraditionalapplicationin Indonesia.
phenol,
flavonoid
and saponin.The effectivedoseof the
Deferoxamineis the only drugof choicefor treatingan
extract
should
be
doubled
to achievethe optimaleffectas
iron overloadin thalasemicpatientsat CiptoMangunkusumo
mangiferinwith theabsorbancevalte 7.22.
Hospital Jakarta.Howeveq not all patientshavethe same
Another advantageis the assumptionthat extractof
toleranceto deferoxamine,renderingpossibility of inadmanggabacangleavesalsohavevitaminC, like theirfruits,l2
equatetherapy.To solve suchproblem, anotherchoice of
drugsuchasdeferasirox,whichhasrecentlybeenapproved but this statementstill needfurther studies.The effect is
patientswith sideeffectsdue
expectedto help thalassemic
bythe FDAand EMEA, is available.Unfortunately,this drug
to the iron overload,increasetheir compliance,capability
is not availablein Indonesiaand is very expensive.To inandqualityof life.
creasetheir effectsfor excretingthe iron complexin kidney,
vitaminC 100mg daily shouldalsobe consumed.a
During
Conclusion
therapy,it is important to asseskidney firnction, such as
Thewaterextractof mangobacangleavesat the dose
creatinineclearance,and that no renal diseaseis found.2,3
1.125mghaschelatingeffecttofenitin serumofthalasemic
In this experiment,deferoxaminecouldnot showtheir
patientsimilarto mangiferinI 00ug in vitro.
absorbance
althoughthe dosewas increased(datanot
shown).Accordingto thisresult,deferoxamine
wasdiscarded
Acknowledgement
from this measurement
or could not be usedfor analysis.
Thanksto DRPM UI who give the fundingfor this reAnother reasonwas that deferoxamineabsorbancecould
Thankyou somuchto all patientsat theDepartment
search.
only be detectedafter specifictreatmentusing expensive
Health FMUI and our medical students:Riska
of
Child
just
chemicalreactionfor 100 samplesand its absorbance
MaelaR. Dewi,DessyFramitaSari,FitriaC.
Wahyuningtyas,
only detectableon lambda440nm.eIn this study,this experiNugrahaeni,
and
Tika Ayu Pratiwi who have fully attended
rrent only had 7 samplesin the positive control group and
research.
this
position on lambda280 nm.
usingthe absorbance
Comparison between absorbancevalue of ferritin before and after
treating with extract of M. foetida Lour.
This exfractefficacy could not be comparedto other
studiesdueto the lack ofdata, exceptin vitro studyusing
mangiferinandfreeiron.5'6Allextractwasshowntheabsorbanceat l, 280 nm, as the fenitin serum,but their optical
324
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