THE EFFECT OF GIVING ETHANOL EXTRACT TO TITHONIA DIVERSIFOLIA LEAVES AS AN ANTIMALARIAL IN BALB/C STRAINS OF MICE BEFORE AND AFTER

INFECTED BY PLASMODIUM BERGHEI

Fiqnanda Ichfal Rizal Medical Faculty of Jember University, Jember, Indonesia. Onset of resistance to antimalarial Plasmodium sp encourages researchers to find new antimalarial to replace an ineffective antimalarial. One attempt to find new antimalarials is through research on medicinal plants traditionally used by people to treat malaria. One of the medicinal plants used as antimalarial is flower month (Tithonia diversifolia (Hemsley) A. Gray) (Calzada and Ciccio, 1995). Kembang bulan (Tithonia diversifolia (Hemley) A. Gray) is a plant species belonging to the Asteraceae family. Herbal plants are reported to have activity as an antimalarial, anti-inflammatory and analgesic (Rungeler et al, 1998). In vivo, ethanol extract of leaf development in proven active against P. berghei at doses of 40; 80; 160; and 320 mg / kg body weight with IC50 values of 114 mg / kg body weight and the IC90 of 475 mg / kg body weight (Budiarti, 2011). Additionally Tithonia diversifolia extract contains substances that can be used as an antimalarial prophylactic and curative in malaria. Then conducted research to determine whether the ethanol extract of leaves of kembang bulan has an influence on the degree of parasitemia of mice strains of Balb / c before and after infected by the parasite Plasmodium berghei (parasitaemia on the degree of mild, moderate and severe). This type of research laboratories is a true experimental design with pretest-posttest randomized control group design. The study design used in this study were randomized pretest-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 as shown 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 after mild (1%), moderate (5%), and severe (10%). Keywords: Tithonia diversifolia, ethanol extract, antimalarial, Plasmodium berghei INTRODUCTION Malaria is an infectious disease caused by Plasmodium sp. These are intracellular parasites, transmitted by female Anopheles mosquito bites. There are four Plasmodium sp that can infect humans, there are: P. falciparum, P. vivax, P. malariae and P. ovale. P. falciparum and P. vivax are the most species found in Indonesia (Depkes RI, 2006). Malaria remains a major health problem in Indonesia especially in eastern Indonesia. There are 433,326 cases of the 232 million population of Indonesia in 2005 (WHO, 2007a) and an explosion of cases of malaria in some areas led to 87 deaths from 18,812 cases in 62 villages in Indonesia in 2005 (WHO, 2007b). By 90% mortality associated with P. falciparum infections

worldwide are caused by severe malaria (Hendrickse, 1997). Magnitude of mortality from severe malaria was likely caused by the ineffectiveness of the drugs given. This happens because in general, patients with malaria were given chloroquine therapy, because chloroquine is the drug of choice for the treatment and prevention of all types of malaria that are used in malaria eradication programs (Depkes RI, 1991). While chloroquine is the drug of choice (first line drug) for treatment of uncomplicated malaria (Tjitra, 2000). Though not all patients who come suffering uncomplicated malaria (malaria mild stage), not infrequently, patients suffering from malaria with complications (severe stage malaria), so that treatment using chloroquine as a first line of this drug becomes ineffective. Various attempts have been made to overcome the various stadium of malaria, among others by avoiding bites of Anopheles mosquitoes, kills adult mosquitoes, kills larvae, reducing the brood, treating people with malaria, vaccinations, and providing preventive treatment, but cases of malaria remains high (Gunawan , 2000). The high incidence of malaria is caused by the emergence of various kinds of obstacles in the eradication of malaria, such as parasite resistance to malaria drugs (Sharif et al, 2006). The emergence of resistance to antimalarial Plasmodium sp encourages researchers to find new antimalarial to replace an ineffective antimalarial. One attempt to find new antimalarials is through research on medicinal plants traditionally used by people to treat malaria. One of the medicinal plants used as antimalarial is flower month (Tithonia diversifolia (Hemsley) A. Gray) (Calzada and Ciccio, 1995).

Kembang bulan (Tithonia diversifolia (Hemley) A. Gray) is a plant species belonging to the Asteraceae family. This plant has long been empirically by the society in South Asia, Central America, and Africa are used to treat some kinds of diseases. In Guatemala, Taiwan, Mexico and Nigeria, the hot water extract of this herb plants used in malaria treatment (Calzada and Ciccio, 1995). Herbal plants are reported to have activity as an antimalarial (Madureira et al., 2002), anti-inflammatory and analgesic (Rungeler et al, 1998). In vivo, ethanol extract of kembang bulan leaf proven active against P. berghei at doses of 40; 80; 160; and 320 mg / kg body weight with IC50 values of 114 mg / kg body weight and the IC90 of 475 mg / kg body weight (Budiarti, 2011). Additionally Tithonia diversifolia extract contains substances that can be used as an antimalarial prophylactic and curative in malaria. This is evidenced by Oyewole et al., (2008) which examined water extracts and methanol extract of Tithonia diversifolia can inhibit the growth of Plasmodium berghei parasites in the blood by 50% when administered before malaria-infected mice at doses 100mg/kg body weight. Based on the above data, the study was conducted to determine whether the ethanol extract of kembang bulan leaves has an influence on the degree of parasitemia of mice strain Balb / c before and after the infected parasite Plasmodium berghei (parasitaemia on the degree of mild, moderate and severe). MATERIALS AND METHODS SAMPLE Male mice strains of Balb / C obtained from the Bagian Ilmu Bahan Alam Fakultas Farmasi, Airlangga University.

PROCEDURE Making Ethanol Extract of Tithonia Diversifolia Leaves that have been sorted then washed, then dried with aerated. Simplicia leaf dry blended and sieved to obtain fine powder. The resulting fine powder and then macerated with technical ethanol solvent as much as 5-10 times the weight of the powder dry for 24 hours while stirring occasionally. The resulting extract was filtered with a funnel to obtain the filtrate. The residue was re-macerated with technical ethanol five times. Concentrating the extract performed with extracts used rotavapor until thickened. Tests on prophylaxis The treated mice were given Suspension of ethanol extract dose 475 mg / kg body weight for 3 days before the inoculation of Plasmodium berghei in intraperitoneal.Suspension of ethanol extract as much as 0.2 mL. After the inoculated test group administered the test material in accordance with the oral dose from day one (H0) until day 5 (H4). The stadium of parasitemia was measured starting H0 to H4. Tests on Various Antimalarial Stadium Antimalarial test conducted at various stadium of in the three groups that had been inoculated P.berghei the degree of parasitemia stadium of mild, moderate, and severe. Suspension extract as much as 0.2 mL of ethanol was prepared at sonde tool to be used. Given test group of test materials in accordance with the dose orally for 3 days (H0 through H4). The degree of parasitemia measured at H0 (after reaching the stadium of of infection mild, moderate and severe) and H4 (after administration of ethanol extract of kembang bulan leaves of 4 days) in accordance with test method antimalarial Peter's Test is modified.

Calculation of percentage of growth and inhibition parasite Thin blood smears were observed number of erythrocytes infected with malaria parasites per 1000 erythrocytes (parasitemia percentage), there are calculated growth percentage and inhibition percentage: % growth = % average growth of prasitemia H4 - % average growth of parasitemi Ho % inhibition = 100% - x 100% Description: Xe: percent average growth of parasites that were given a certain dose of test material Xk: percentage of parasite growth at an average of negative control Data Analysis Test for normality with kormogorov Smirnov test is performed to determine whether or not the normal distribution of data. The analysis used Oneway ANOVA test was continued LSD. The results are then presented in tabular form and discussed in narrative form. Data processing using the help of SPSS Statistics 19.0 software. RESULTS Research results obtained by ethanol extract of kembang bulan leaves dose of 475 mg / kg body weight into the degree of parasitemia of mice Balb / c infected by Plasmodium berghei before and at various stadium of of infection (mild, moderate and severe) as follows: Prophylaxis group had a number of degrees of parasitemia lower than the negative control group (Picture 1). The group of mild stadium of has a number of degrees of parasitemia lower than the negative control group (Picture 2).

The group of moderate stadium of has a number of degrees of parasitemia lower than the negative control group (Picture 3). The group of severe stadium of has a number of degrees of parasitemia lower than the negative control group (Picture 4). Based on data from the various percentages of inhibition of extract ethanol of kembang bulan leaf for growth in P. berghei before infected and at various stadium of infection (mild, moderate, and severe), can be seen that in the prophylaxis group had the greatest percentage of inhibition (Picture 5). Based on the data obtained is then performed to test for normality using Kolmogorov-Smirnov test to see whether the obtained data are normally distributed or not. The results obtained normality test of significance p> 0.05 which shows the data from each group are normally distributed (table 1). Therefore the data are normally distributed parametric test used by Oneway ANOVA test to compare the effectiveness of the influence of ethanol extract of kembang bulan leaf in both as prophylaxis before the inoculation of Plasmodium berghei as well as an antimalarial in various stadium of malaria. Based on the analysis of data obtained with One-way ANOVA significance level of 0.000 (sig <0.05), meaning that there are significant differences barriers parasitemia by ethanol extract of kembang bulan leaf (table 2). Then followed LSD test to determine the barriers parasitemia by ethanol extract of kembang bulan leaf between treatment groups with each other. Confidence level used is 95% which means if the obtained significance value <0.05 then there are significant differences between the treatment compared to other treatments. Based on the results of LSD test inhibition parasitemia in the prophylactic treatment group compared with treated

group the on the stadium of mild and moderate obtained significance value p = 0.000 (<0.05). When compared with treatment groups at the severe stadium obtain of the value of significance p = 0.002 (<0.05), this means that there are significant differences between treatment groups with the prophylactic treatment group on the stage of mild, moderate, and severe (Table 1). In the mild stadium of the treatment group compared with the moderate stadium was obtained significance value of p = 0.112, this means that there was no significant difference between the mild and moderate stadium. When compared with the severe stadium was obtained significance value of p = 0.003, this means that there are significant differences between the stadium of mild and severe stadium (Table 1). In the moderate stadium of the treatment group compared with the severe stadium was obtained significance value of p = 0.036, this means that there are significant differences between the stadium of mild and severe stadium (Table 1). DISCUSSION Test effect of ethanol extract of kembang bulan leaves into the degree of parasitemia before and after infected by Plasmodium berghei at different stadium of infection as mild, moderate and severe, aims to see the potential of kembang bulan leaves as prophylactic and curative malaria that can be used at various stadium of infection. From the analysis of research data found that the ethanol extract of kembang bulan leaves proved to have an influence on the stadium of parasitemia before and after by Plasmodium berghei infected at the stadium of mild, moderate, and severe. This is appropriate with the hypothesis that the ethanol extract of kembang bulan leaves has a prophylactic effect in mice infected with by Plasmodium berghei and antimalarial

therapy in mice infected with by Plasmodium berghei stadium of mild, moderate and severe. Based on research data that percentage of stadium of parasitaemia in the prophylactic group, that groups of mice that had been given ethanol extract of kembang bulan leaves dose of 475 mg / kg body weight three days before infected with by Plasmodium berghei, indicating a lower number than the negative control group. This means that ethanol extracts of kembang bulan leaves flower moon have an influence as malaria prophylaxis with a power resistor to the development of the parasite. The results are consistent with Oyewole et al, 2008 stating that exstracts water and methanol extracts of kembang bulan leaves development in more effective when administered before the onset of infection. Based on the results of research in the treatment group the various stadium of infection that mild, moderate and severe stadium, the ethanol extract of kembang bulan leaves dose of 475 mg / kg body weight showed an average stadium of parasitemia lower than the control group. This means that ethanol extracts have an influence as antimalarials at various stadium of infection by suppressing the growth of the parasite, although not one hundred percent kill the parasite. The results of this study support previous research that showed that administration of methanol extract and water extract of kembang bulan leaves can reduce stadium of parasitemia and extend the life of mice compared to control group, but can not prevent mortality from severe anemia (Oyewole et al, 2008). Kembang bulan (Tithonia diversifolia) leaves proved to have an influence on the degree of parasitemia in Plasmodium berghei infected mice before and after the infected by Plasmodium berghei at various stadium of malaria (1%, 5%, 10%). The ability of kembang bulan extracts as

antiplasmodium because it contains an active ingredient called sesquiterpene lactone taginin C that have been shown to kill Plasmodium falciparum strain of FCA (IC 50: 0.75 microg / mL) (Goffin et al in Titanji et al, 2008). Afiyah (2007) states that the ether fraction of methanol extract of kembang bulan leaves has antiplasmodium activity in P. falciparum strain FCR-3 in vitro by inhibiting the polymerization of heme. The ability of a antiplasmodium in inhibiting heme polymerization takes place with his ability as an antimalarial, although it is known that the mechanism of action antiplasmodium not only through inhibition of heme. Heme polymerization inhibitory activity is actually a working one or two mechanisms, there are (1) there is interaction between terpenoid compounds, phenols and sterols with heme elektrolik system, (2) These extracts consist of compounds that have hydroxyl groups that can bind to heme iron ions (Bassilico, et al., 1998; Sharif, 2007). Goffin et al., (2002) reported that the kembang bulan extracts in vitro is able to fight three strains of P. falciparum in which the ether extract of this herb plant has the best antiplasmodium activity with IC50 of 0.75 g / mL. The results of this ether extract fractionation, found the lactone seskuiterpen taginin C which is the active component against Plasmodium. The results of statistical analysis of percentage inhibition (percentage inhibition of kembang bulan extract on the growth of Plasmodium berghei) by using Oneway ANOVA showed differences between groups prophylactic treatment and stadium infection of treatment group (mild, moderate and severe). The results of further analysis using LSD test found a significant difference between groups prophylactic treatment and group with the various stadium of infection (mild, moderate and severe). And between treatment groups of mild stadium and

moderate stadium with a severe stadium infection. However, in the treated group of a mild stadium infection with moderate stadium there was no significant difference. This shows that the ethanol extract of kembang bulan leaves contains of Antimalarial materials that can provide preventive and curative effect against malaria infection. However, ethanol extract of kembang bulan leaf before the infection (as prophylaxis) gives better results compared with administration during infection. Therefore it can be concluded that the ethanol extract of kembang bulan leaves has an influence on the degree of parasitemia before infected and after infected by Plasmodium berghei of various stadium of malaria (mild, moderate, severe). This is appropriate with previous studies showing that both methanol extract and water extract of kembang bulan leaves contain of antimalarial substances with properties that indicate both preventive and curative effects on malaria parasites (Oyewole et al, 2008). However, the ability of ethanol extract of kembang bulan leaf to inhibit the growth depends on the time of administration. This is approproate with research Oyewole et al, 2008, which proves that mice given the methanol extract and water extracts of infected leaves before the flower-month growth have lower parasitemia and survival (survival) is better than the group of mice who were treated after the infection.

AVERAGE PERCENTAGE OF PARASITAEMIA DEGREES

0,35 % Parasitaenia Degrees 0,3 0,2 0,15 0,1 0,05 0 H0 H1 H2 H3 H4 % Parasitemiae degrees 0,25 Negative control Prophylaxis 30 25 20 15 10 5 0

AVERAGE PERCENTAGE OF PARASITAEMIA DEGREES

Negative control Severe Stadium

Picture 1. The average degree of parasitemia Prophylaxis group and Control Negative Group
AVERAGE PERCENTAGE OF PARASITAEMIA DEGREES
20 18 16 14 12 10 8 6 4 2 0 H0 H1 H2 H3 H4 % Parasitaemia Degrees

H0

H1

H2

H3

H4

Picture 4. The average degree of parasitemia Severe Stadium Group and Control Negative Group
AVERAGE PERCENTAGE OF GROWTH AND INHIBITION

Negative control Mild Stadium

Picture 2. The average degree of parasitemia Mild Stadium Group and Control Negative Group
AVERAGE PERCENTAGE OF PARASITAEMIA DEGREES
20 18 16 14 12 10 8 6 4 2 0 H0 H1 H2 H3 H4 % PArasitaemia Degrees

100 90 80 70 60 50 40 30 20 10 0
PROPHYLAXIS MILD STADIUM MODERATE STADIUM SEVERE STADIUM

Percentage

AVERAGE PERCENTAGE OF NEGATIVE CONTROL GROWTH AVERAGE PERCENTAGE OF TREATMENT GROUP GROWTH AVERAGE PERCENTAGE OF INHIBITION

Negative Control Moderate stadium

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

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

Table 1. Normality Kolmogorov-smirnov Test Results c. a. Prophylaxis Group

One-Sample Kolmogorov-Smirnov Test Parasitaemia Inhibition N Normal Parametersa,b Most Extreme Differences Kolmogorov-Smirnov Z Asymp. Sig. (2-tailed) a. Test distribution is Normal. b. Calculated from data. Mean Std. Deviation Absolute Positive Negative 3 100,0000 ,01000 ,175 ,175 -,175 ,303 1,000

Moderate Stadium Group

One-Sample Kolmogorov-Smirnov Test Parasitaemia Inhibition N 3 Normal Parametersa,b Mean 56,8400 Std. Deviation 5,88062 Most Extreme Differences Absolute ,358 Positive ,358 Negative -,257 Kolmogorov-Smirnov Z ,619 Asymp. Sig. (2-tailed) ,838 a. Test distribution is Normal. b. Calculated from data.

d.

Severe Stadium Group

b.

Mild Stadium Group

One-Sample Kolmogorov-Smirnov Test Parasitaemia Inhibition

N Normal Parametersa,b

Most Extreme Differences

Mean Std. Deviation Absolute Positive Negative

3 45,6900 9,79062 ,340 ,243 -,340 ,590 ,878

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

One-Sample Kolmogorov-Smirnov Test Parasitaemia Inhibition N 3 Normal Parametersa,b Mean 72,6133 Std. Deviation 10,24415 Most Extreme Differences Absolute ,374 Positive ,272 Negative -,374 Kolmogorov-Smirnov Z ,648 Asymp. Sig. (2-tailed) ,795 a. Test distribution is Normal. b. Calculated from data.

Table 2. Way Anova test Results

Sum of Squares Between Groups Within Groups Total 4992,511 470,248 5462,759 df 3 8 11 Mean Square F Sig. ,000

Table 3. LSD Test Results

(I) Kelompok 0 (J) Kelompok 1 Mean Differenc e (I-J) 54,30667 95% Confidence Interval Std. Error 6,25998 Sig. ,000 Lower Bound 39,8711 Upper Bound
68,7422

1664,170 28,311 58,781

43,12667

6,25998

,000

28,6911

57,5622

10

27,38333

6,25998

,002

12,9478

41,8189

54,30667
*

6,25998

,000

-68,7422

-39,8711

11,18000 26,92333
*

6,25998

,112

-25,6155

3,2555

10

6,25998

,003

-41,3589

-12,4878

43,12667
*

6,25998

,000

-57,5622

-28,6911

1 10

11,18000 15,74333
*

6,25998 6,25998

,112 ,036

-3,2555 -30,1789

25,6155 -1,3078

10

27,38333
*

6,25998

,002

-41,8189

-12,9478

26,92333

6,25998

,003

12,4878

41,3589

15,74333

6,25998

,036

1,3078

30,1789

(I) Kelompok 0

(J) Kelompok 1

Mean Differenc e (I-J) 54,30667

95% Confidence Interval Std. Error 6,25998 Sig. ,000 Lower Bound 39,8711 Upper Bound
68,7422

43,12667

6,25998

,000

28,6911

57,5622

10

27,38333

6,25998

,002

12,9478

41,8189

54,30667
*

6,25998

,000

-68,7422

-39,8711

11,18000 26,92333
*

6,25998

,112

-25,6155

3,2555

10

6,25998

,003

-41,3589

-12,4878

43,12667
*

6,25998

,000

-57,5622

-28,6911

1 10

11,18000 15,74333
*

6,25998 6,25998

,112 ,036

-3,2555 -30,1789

25,6155 -1,3078

10

27,38333
*

6,25998

,002

-41,8189

-12,9478

26,92333

6,25998

,003

12,4878

41,3589

15,74333

6,25998

,036

1,3078

30,1789

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

REFERENCES Basilico, N., Pagani, E., Monti, D., Olliaro, P., dan Taramelli, D. 1998. A Microtirebased Method for Measuring the Haem Polymerization Inhibitory Avtivity (HPIA) of Antimaralial Drugs. Journal of Antimicrobial Chemotheraphy, 42: 55-60 Calzada, J. G., & Ciccio, J. F. 1995. Aislamiento de Tirotundina a partir de Tithonia diversifolia (Hemsl.) Gray. Rev. Latinoamer. Quim. 9: 202-203. Departemen Kesehatan Republik Indonesia, Direktorat Jenderal Pemberantasan Penyakit Menular dan Penyehatan Lingkungan Pemukiman. Malaria : Pengobatan no 3. 1991. Jakarta Departemen Kesehatan RI., 2006. Pedoman Penatalaksanaan Kasus Malaria di Indonesia. Jakarta: Direktorat Jendral Pengendalian Penyakit dan Penyehatan lingkungan Goffin, E., Ziemons, E., De Mol, P., Do Ceu De Madureina, dan M., Martins. 2002. In vitro antiplasmodial avtivity of Tithonia diversifolia and identification of its main active constituent: Tagitinin C. Planta Med. 68 (6): 543-545. Gunawan, S. 2000. Epidemiologi Malaria (in) P.N. Harijanto. (ed): Malaria, Epidemiologi, Patogenesis, Manifestasi Klinis dan Penanganan. EGC. Penerbit Buku Kedokteran. Jakarta.1-13. Hendrickse, R.G. Malaria and Child Health. 1997. Annals of Tropical Medicine and Parasitology. 81:503. Madureira, M. C., Martins, A. P., Gomes, M., Paiva, J., Cunha, A. P., dan Rosario, V. 2002. Antimalarial activity of medicinal plants used in traditional medicine in S. Tome and Principe islands. J. Ethnopharmacol. 81: 23-29. Oyewole, L. O., Ibidapo, C. A., Moronkola, D. O., Oduola, A. O., Adeoye, G. O., Anyasor, G. N., dan Obansa, J. A. 2008.

Anti-malarial and repellent activities of Tithonia diversifolia (Hemsl.) leaf extracts. Journal of Medicinal Plants Research Vol. 2 (8), pp. 171-175 Rungeler, P., Lyss, G., Castro, V., Mora, G., Pahl, H.L., Merfort, I. 1998. Study of three sesquiterpene lactones from Tithonia diversifolia on their antiinflammatory activity using the transcription factor NF-kappa B and enzyme of the arachidonic acid pathway as target. Planta med. 64 588-593. Syarif, R. A., Wahyuningsih, M. S. A., Ngatidjan, M., Kurniawan, H., dan Hilal, S. R. A. 2006. Aktivitas Antiplasmodium In Vitro Ekstrak Kembang Bulan (Tithonia diversifolia (Hemsley) A.Gray)) Terhadap Plasmodium falciparum. Tesis Penelitian Bagian Farmasi Kedokteran dan Bagian Farmakologi dan Toksikologi Fakultas Kedokteran Universitas Gadjah Mada. Tjitra, E. 2000. Obat-obat malaria, Dalam: harijanto PN (editor) Malaria, Epidemologi, Patogenesis, Manifestasi Klinis, & Penanganan. Jakarta: Penerbit Buku Kedoktean EGC. 194223 WHO. 2007a. Malaria Situation in SEAR Countries : Indonesia in Malaria. WHO. Regional Office for South-East Asia WHO. 2007b. Malaria Epidemics / Outbreaks in SEA Region in Malaria. WHO. Regional Office for South-East Asia.