Jurnal Ekstrak Etanol Daun Kembang Bulan Bing(1)

THE EFFECT OF GIVING ETHANOL EXTRACT TO TITHONIADIVERSIFOLIA LEAVES AS AN ANTIMALARIAL IN BALB/C STRAINS

OF MICE BEFORE AND AFTER INFECTED BY PLASMODIUMBERGHEI

Fiqnanda Ichfal Rizal

Medical Faculty of Jember University, Jember, Indonesia.

Onset of resistance to antimalarial Plasmodium sp encourages researchers to findnew antimalarial to replace an ineffective antimalarial. One attempt to find newantimalarials is through research on medicinal plants traditionally used by people to treatmalaria. One of the medicinal plants used as antimalarial is flower month (Tithoniadiversifolia (Hemsley) A. Gray) (Calzada and Ciccio, 1995).

Kembang bulan (Tithonia diversifolia (Hemley) A. Gray) is a plant speciesbelonging to the Asteraceae family. Herbal plants are reported to have activity as anantimalarial, anti-inflammatory and analgesic (Rungeler et al, 1998). In vivo, ethanolextract of leaf development in proven active against P. berghei at doses of 40; 80; 160; and320 mg / kg body weight with IC50 values of 114 mg / kg body weight and the IC90 of 475mg / kg body weight (Budiarti, 2011). Additionally Tithonia diversifolia extract containssubstances that can be used as an antimalarial prophylactic and curative in malaria. Thenconducted research to determine whether the ethanol extract of leaves of kembang bulanhas an influence on the degree of parasitemia of mice strains of Balb / c before and afterinfected by the parasite Plasmodium berghei (parasitaemia on the degree of mild, moderateand severe).

This type of research laboratories is a true experimental design with pretest-posttestrandomized control group design. The study design used in this study were randomizedpretest-posttest control group design, with 4 treatment groups (prophylaxis, mild,moderate and severe) and a negative control group.

Based on this research and development in the ethanol extract of the leaves asshown to have antimalarial activity in mice Balb / c before inoculated Plasmodium berghei(prophylaxis) and as an antimalarial in mice Balb / c infected by malaria at a stage aftermild (1%), moderate (5%), and severe (10%).

Keywords: Tithonia diversifolia, ethanol extract, antimalarial, Plasmodium berghei

INTRODUCTIONMalaria is an infectious disease

caused by Plasmodium sp. These areintracellular parasites, transmitted by femaleAnopheles mosquito bites. There are fourPlasmodium sp that can infect humans, thereare: P. falciparum, P. vivax, P. malariae andP. ovale. P. falciparum and P. vivax are themost species found in Indonesia (Depkes RI,2006).

Malaria remains a major healthproblem in Indonesia especially in easternIndonesia. There are 433,326 cases of the232 million population of Indonesia in 2005(WHO, 2007a) and an explosion of cases ofmalaria in some areas led to 87 deaths from18,812 cases in 62 villages in Indonesia in2005 (WHO, 2007b). By 90% mortalityassociated with P. falciparum infections

worldwide are caused by severe malaria(Hendrickse, 1997).

Magnitude of mortality from severemalaria was likely caused by theineffectiveness of the drugs given. Thishappens because in general, patients withmalaria were given chloroquine therapy,because chloroquine is the drug of choicefor the treatment and prevention of all typesof malaria that are used in malariaeradication programs (Depkes RI, 1991).While chloroquine is the drug of choice(first line drug) for treatment ofuncomplicated malaria (Tjitra, 2000).Though not all patients who come sufferinguncomplicated malaria (malaria mild stage),not infrequently, patients suffering frommalaria with complications (severe stagemalaria), so that treatment using chloroquineas a first line of this drug becomesineffective.

Various attempts have been made toovercome the various stadium of malaria,among others by avoiding bites ofAnopheles mosquitoes, kills adultmosquitoes, kills larvae, reducing the brood,treating people with malaria, vaccinations,and providing preventive treatment, butcases of malaria remains high (Gunawan ,2000). The high incidence of malaria iscaused by the emergence of various kinds ofobstacles in the eradication of malaria, suchas parasite resistance to malaria drugs(Sharif et al, 2006).

The emergence of resistance toantimalarial Plasmodium sp encouragesresearchers to find new antimalarial toreplace an ineffective antimalarial. Oneattempt to find new antimalarials is throughresearch on medicinal plants traditionallyused by people to treat malaria. One of themedicinal plants used as antimalarial isflower month (Tithonia diversifolia(Hemsley) A. Gray) (Calzada and Ciccio,1995).

Kembang bulan (Tithoniadiversifolia (Hemley) A. Gray) is a plantspecies belonging to the Asteraceae family.This plant has long been empirically by thesociety in South Asia, Central America, andAfrica are used to treat some kinds ofdiseases. In Guatemala, Taiwan, Mexico andNigeria, the hot water extract of this herbplants used in malaria treatment (Calzadaand Ciccio, 1995). Herbal plants arereported to have activity as an antimalarial(Madureira et al., 2002), anti-inflammatoryand analgesic (Rungeler et al, 1998).

In vivo, ethanol extract of kembangbulan leaf proven active against P. berghei atdoses of 40; 80; 160; and 320 mg / kg bodyweight with IC50 values of 114 mg / kgbody weight and the IC90 of 475 mg / kgbody weight (Budiarti, 2011). AdditionallyTithonia diversifolia extract containssubstances that can be used as anantimalarial prophylactic and curative inmalaria. This is evidenced by Oyewole etal., (2008) which examined water extractsand methanol extract of Tithonia diversifoliacan inhibit the growth of Plasmodiumberghei parasites in the blood by 50% whenadministered before malaria-infected mice atdoses 100mg/kg body weight.

Based on the above data, the studywas conducted to determine whether theethanol extract of kembang bulan leaves hasan influence on the degree of parasitemia ofmice strain Balb / c before and after theinfected parasite Plasmodium berghei(parasitaemia on the degree of mild,moderate and severe).

MATERIALS AND METHODS

SAMPLE

Male mice strains of Balb / C obtainedfrom the Bagian Ilmu Bahan Alam FakultasFarmasi, Airlangga University.

PROCEDURE

Making Ethanol Extract of TithoniaDiversifolia

Leaves that have been sorted thenwashed, then dried with aerated. Simplicialeaf dry blended and sieved to obtain finepowder. The resulting fine powder and thenmacerated with technical ethanol solvent asmuch as 5-10 times the weight of thepowder dry for 24 hours while stirringoccasionally. The resulting extract wasfiltered with a funnel to obtain the filtrate.The residue was re-macerated with technicalethanol five times. Concentrating the extractperformed with extracts used rotavapor untilthickened.

Tests on prophylaxisThe treated mice were given

Suspension of ethanol extract dose 475 mg /kg body weight for 3 days before theinoculation of Plasmodium berghei inintraperitoneal.Suspension of ethanol extractas much as 0.2 mL. After the inoculated testgroup administered the test material inaccordance with the oral dose from day one(H0) until day 5 (H4). The stadium ofparasitemia was measured starting H0 to H4.

Tests on Various Antimalarial StadiumAntimalarial test conducted at various

stadium of in the three groups that had beeninoculated P.berghei the degree ofparasitemia stadium of mild, moderate, andsevere. Suspension extract as much as 0.2mL of ethanol was prepared at sonde tool tobe used. Given test group of test materials inaccordance with the dose orally for 3 days(H0 through H4). The degree of parasitemiameasured at H0 (after reaching the stadiumof of infection mild, moderate and severe)and H4 (after administration of ethanolextract of kembang bulan leaves of 4 days)in accordance with test method antimalarialPeter's Test is modified.

Calculation of percentage of growth andinhibition parasite

Thin blood smears were observednumber of erythrocytes infected withmalaria parasites per 1000 erythrocytes(parasitemia percentage), there arecalculated growth percentage and inhibitionpercentage:% growth = % average growth of

prasitemia H4 - % averagegrowth of parasitemi Ho

% inhibition = 100% - x 100%

Description:Xe: percent average growth of parasites thatwere given a certain dose of test materialXk: percentage of parasite growth at anaverage of negative control

Data AnalysisTest for normality with kormogorov -

Smirnov test is performed to determinewhether or not the normal distribution ofdata. The analysis used Oneway ANOVAtest was continued LSD. The results are thenpresented in tabular form and discussed innarrative form. Data processing using thehelp of SPSS Statistics 19.0 software.

RESULTSResearch results obtained by ethanol

extract of kembang bulan leaves dose of 475mg / kg body weight into the degree ofparasitemia of mice Balb / c infected byPlasmodium berghei before and at variousstadium of of infection (mild, moderate andsevere) as follows:

Prophylaxis group had a number ofdegrees of parasitemia lower than thenegative control group (Picture 1).

The group of mild stadium of has anumber of degrees of parasitemia lower thanthe negative control group (Picture 2).

The group of moderate stadium of has anumber of degrees of parasitemia lower thanthe negative control group (Picture 3).

The group of severe stadium of has anumber of degrees of parasitemia lower thanthe negative control group (Picture 4).

Based on data from the variouspercentages of inhibition of extract ethanolof kembang bulan leaf for growth in P.berghei before infected and at variousstadium of infection (mild, moderate, andsevere), can be seen that in the prophylaxisgroup had the greatest percentage ofinhibition (Picture 5).

Based on the data obtained is thenperformed to test for normality usingKolmogorov-Smirnov test to see whetherthe obtained data are normally distributed ornot. The results obtained normality test ofsignificance p> 0.05 which shows the datafrom each group are normally distributed(table 1).

Therefore the data are normallydistributed parametric test used by OnewayANOVA test to compare the effectiveness ofthe influence of ethanol extract of kembangbulan leaf in both as prophylaxis before theinoculation of Plasmodium berghei as wellas an antimalarial in various stadium ofmalaria. Based on the analysis of dataobtained with One-way ANOVAsignificance level of 0.000 (sig <0.05),meaning that there are significantdifferences barriers parasitemia by ethanolextract of kembang bulan leaf (table 2).

Then followed LSD test to determinethe barriers parasitemia by ethanol extract ofkembang bulan leaf between treatmentgroups with each other. Confidence levelused is 95% which means if the obtainedsignificance value <0.05 then there aresignificant differences between the treatmentcompared to other treatments.

Based on the results of LSD testinhibition parasitemia in the prophylactictreatment group compared with treated

group the on the stadium of mild andmoderate obtained significance value p =0.000 (<0.05). When compared withtreatment groups at the severe stadiumobtain of the value of significance p = 0.002(<0.05), this means that there are significantdifferences between treatment groups withthe prophylactic treatment group on thestage of mild, moderate, and severe (Table1).

In the mild stadium of the treatmentgroup compared with the moderate stadiumwas obtained significance value of p =0.112, this means that there was nosignificant difference between the mild andmoderate stadium. When compared with thesevere stadium was obtained significancevalue of p = 0.003, this means that there aresignificant differences between the stadiumof mild and severe stadium (Table 1).

In the moderate stadium of thetreatment group compared with the severestadium was obtained significance value of p= 0.036, this means that there are significantdifferences between the stadium of mild andsevere stadium (Table 1).

DISCUSSIONTest effect of ethanol extract of

kembang bulan leaves into the degree ofparasitemia before and after infected byPlasmodium berghei at different stadium ofinfection as mild, moderate and severe, aimsto see the potential of kembang bulan leavesas prophylactic and curative malaria that canbe used at various stadium of infection.From the analysis of research data foundthat the ethanol extract of kembang bulanleaves proved to have an influence on thestadium of parasitemia before and after byPlasmodium berghei infected at the stadiumof mild, moderate, and severe. This isappropriate with the hypothesis that theethanol extract of kembang bulan leaves hasa prophylactic effect in mice infected withby Plasmodium berghei and antimalarial

therapy in mice infected with byPlasmodium berghei stadium of mild,moderate and severe.

Based on research data that percentageof stadium of parasitaemia in theprophylactic group, that groups of mice thathad been given ethanol extract of kembangbulan leaves dose of 475 mg / kg bodyweight three days before infected with byPlasmodium berghei, indicating a lowernumber than the negative control group.This means that ethanol extracts of kembangbulan leaves flower moon have an influenceas malaria prophylaxis with a power resistorto the development of the parasite. Theresults are consistent with Oyewole et al,2008 stating that exstracts water andmethanol extracts of kembang bulan leavesdevelopment in more effective whenadministered before the onset of infection.

Based on the results of research in thetreatment group the various stadium ofinfection that mild, moderate and severestadium, the ethanol extract of kembangbulan leaves dose of 475 mg / kg bodyweight showed an average stadium ofparasitemia lower than the control group.This means that ethanol extracts have aninfluence as antimalarials at various stadiumof infection by suppressing the growth of theparasite, although not one hundred percentkill the parasite. The results of this studysupport previous research that showed thatadministration of methanol extract and waterextract of kembang bulan leaves can reducestadium of parasitemia and extend the life ofmice compared to control group, but can notprevent mortality from severe anemia(Oyewole et al, 2008).

Kembang bulan (Tithonia diversifolia)leaves proved to have an influence on thedegree of parasitemia in Plasmodiumberghei infected mice before and after theinfected by Plasmodium berghei at variousstadium of malaria (1%, 5%, 10%). Theability of kembang bulan extracts as

antiplasmodium because it contains anactive ingredient called sesquiterpenelactone taginin C that have been shown tokill Plasmodium falciparum strain of FCA(IC 50: 0.75 microg / mL) (Goffin et al inTitanji et al, 2008). Afiyah (2007) states thatthe ether fraction of methanol extract ofkembang bulan leaves has antiplasmodiumactivity in P. falciparum strain FCR-3 invitro by inhibiting the polymerization ofheme. The ability of a antiplasmodium ininhibiting heme polymerization takes placewith his ability as an antimalarial, althoughit is known that the mechanism of actionantiplasmodium not only through inhibitionof heme. Heme polymerization inhibitoryactivity is actually a working one or twomechanisms, there are (1) there isinteraction between terpenoid compounds,phenols and sterols with heme elektroliksystem, (2) These extracts consist ofcompounds that have hydroxyl groups thatcan bind to heme iron ions (Bassilico, et al.,1998; Sharif, 2007). Goffin et al., (2002)reported that the kembang bulan extracts invitro is able to fight three strains of P.falciparum in which the ether extract of thisherb plant has the best antiplasmodiumactivity with IC50 of 0.75 μg / mL. Theresults of this ether extract fractionation,found the lactone seskuiterpen taginin Cwhich is the active component againstPlasmodium.

The results of statistical analysis ofpercentage inhibition (percentage inhibitionof kembang bulan extract on the growth ofPlasmodium berghei) by using OnewayANOVA showed differences betweengroups prophylactic treatment and stadiuminfection of treatment group (mild, moderateand severe). The results of further analysisusing LSD test found a significant differencebetween groups prophylactic treatment andgroup with the various stadium of infection(mild, moderate and severe). And betweentreatment groups of mild stadium and

moderate stadium with a severe stadiuminfection. However, in the treated group of amild stadium infection with moderatestadium there was no significant difference.This shows that the ethanol extract ofkembang bulan leaves contains ofAntimalarial materials that can providepreventive and curative effect againstmalaria infection. However, ethanol extractof kembang bulan leaf before the infection(as prophylaxis) gives better resultscompared with administration duringinfection.

Therefore it can be concluded that theethanol extract of kembang bulan leaves hasan influence on the degree of parasitemiabefore infected and after infected byPlasmodium berghei of various stadium ofmalaria (mild, moderate, severe). This isappropriate with previous studies showingthat both methanol extract and water extractof kembang bulan leaves contain ofantimalarial substances with properties thatindicate both preventive and curative effectson malaria parasites (Oyewole et al, 2008).However, the ability of ethanol extract ofkembang bulan leaf to inhibit the growthdepends on the time of administration. Thisis approproate with research Oyewole et al,2008, which proves that mice given themethanol extract and water extracts ofinfected leaves before the flower-monthgrowth have lower parasitemia and survival(survival) is better than the group of micewho were treated after the infection.

Picture 1. The average degree of parasitemia Prophylaxis group and ControlNegative Group

Picture 2. The average degree of parasitemia Mild Stadium Group and ControlNegative Group

Picture 3. The average degree of parasitemia Moderate Stadium Group andControl Negative Group

Picture 4. The average degree of parasitemia Severe Stadium Group and ControlNegative Group

Picture 5. The average of percentage growth and the average percentageinhibition of the ethanol extract of kembang bulan leaf on the growth P. bergheibefore infected and at various stadium of infection (mild, moderate, and severe)

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

H0 H1 H2 H3 H4

% P

aras

itaen

ia D

egre

es

AVERAGE PERCENTAGE OF PARASITAEMIA DEGREES

Negative control

Prophylaxis

02468

101214161820

H0 H1 H2 H3 H4

% P

aras

itaem

ia D

egre

es


Negative control

Mild Stadium

02468

101214161820

H0 H1 H2 H3 H4

% P

Ara

sitae

mia

Deg

rees


Negative Control

Moderate stadium

0

5

10

15

20

25

30

H0 H1 H2 H3 H4

% P

aras

item

iae

degr

ees


Negative control

Severe Stadium

0102030405060708090

100

PROPHYLAXIS MILD STADIUM MODERATESTADIUM

SEVERE STADIUM

Perc

enta

ge

AVERAGE PERCENTAGE OF GROWTH AND INHIBITION

AVERAGE PERCENTAGEOF NEGATIVE CONTROLGROWTH

AVERAGE PERCENTAGEOF TREATMENT GROUPGROWTH

AVERAGE PERCENTAGEOF INHIBITION

Table 1. Normality Kolmogorov-smirnov Test Results

a. Prophylaxis GroupOne-Sample Kolmogorov-Smirnov Test

Parasitaemia InhibitionN 3Normal Parametersa,b Mean 100,0000

Std.Deviation

,01000

Most Extreme Differences Absolute ,175Positive ,175Negative -,175

Kolmogorov-Smirnov Z ,303Asymp. Sig. (2-tailed) 1,000a. Test distribution is Normal.b. Calculated from data.

b. Mild Stadium Group

One-Sample Kolmogorov-Smirnov TestParasitaemia Inhibition

N 3Normal Parametersa,b Mean 45,6900

Std.Deviation

9,79062

Most Extreme Differences Absolute ,340Positive ,243Negative -,340

Kolmogorov-Smirnov Z ,590Asymp. Sig. (2-tailed) ,878a. Test distribution is Normal.b. Calculated from data.

c. Moderate Stadium Group



Std. Deviation 5,88062Most Extreme Differences Absolute ,358

Positive ,358Negative -,257


d. Severe Stadium Group



Std. Deviation 10,24415Most Extreme Differences Absolute ,374

Positive ,272Negative -,374


Table 2. Way Anova test Results

Sum ofSquares df

MeanSquare F Sig.

BetweenGroups

4992,511 3 1664,170 28,311 ,000

WithinGroups

470,248 8 58,781

Total 5462,759 11

Table 3. LSD Test Results

(I)Kelompok

(J)Kelompok

MeanDifferenc

e (I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

0 1 54,30667*

6,25998 ,000 39,8711 68,7422

5 43,12667*

6,25998 ,000 28,6911 57,5622

10 27,38333*

6,25998 ,002 12,9478 41,8189

1 0 -54,30667

*

6,25998 ,000 -68,7422 -39,8711

5 -11,18000

6,25998 ,112 -25,6155 3,2555

10 -26,92333

*

6,25998 ,003 -41,3589 -12,4878

0 -43,12667

*

6,25998 ,000 -57,5622 -28,6911

1 11,18000 6,25998 ,112 -3,2555 25,6155

10 -15,74333

*

6,25998 ,036 -30,1789 -1,3078

10 0 -27,38333

*

6,25998 ,002 -41,8189 -12,9478

1 26,92333*

6,25998 ,003 12,4878 41,3589

5 15,74333*

6,25998 ,036 1,3078 30,1789

(I)Kelompok

(J)Kelompok

MeanDifferenc

e (I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

0 1 54,30667*

6,25998 ,000 39,8711 68,7422

5 43,12667*

6,25998 ,000 28,6911 57,5622

10 27,38333*

6,25998 ,002 12,9478 41,8189

1 0 -54,30667

*

6,25998 ,000 -68,7422 -39,8711

5 -11,18000

6,25998 ,112 -25,6155 3,2555

10 -26,92333

*

6,25998 ,003 -41,3589 -12,4878

0 -43,12667

*

6,25998 ,000 -57,5622 -28,6911

1 11,18000 6,25998 ,112 -3,2555 25,6155

10 -15,74333

*

6,25998 ,036 -30,1789 -1,3078

10 0 -27,38333

*

6,25998 ,002 -41,8189 -12,9478

1 26,92333*

6,25998 ,003 12,4878 41,3589

5 15,74333*

6,25998 ,036 1,3078 30,1789

*. The mean difference is significant at the 0.05 level.

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Jurnal Ekstrak Etanol Daun Kembang Bulan Bing(1)