Stage Specificity of Eurycomanone Isolated from Eurycoma longifolia on Plasmodium falciparum Cycles

1Department of Pharmacology and Therapy, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia 2Department of Parasitology, Faculty of Medicine, Universitas Gadjah Mada Yogyakarta, Indonesia 3Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia 4Faculty of Medicine, University of Jenderal Soedirman, Purwokerto, Indonesia 5Department of Pharmacology and Therapy, Faculty of Medicine, University of Syiah Kuala, Banda Aceh, Indonesia


Introduction
Malaria is still health problem in the world. Globally, an estimated 3.3 billion people in 97 countries and territories are at risk of being infected with malaria and developing disease and 1.2 billion are at high risk (WHO, 2014). The increasing resistance of Plasmodium falciparum strains to currently available anti-malarial has initiated numerous studies aimed at identifying new antimalarial agents. One of the strategies in search for new anti-malarial compounds is a research of active plant constituents. Medicinal plants have been used traditionally to treat of malaria in some countries in the world. Significant success was achieved with the new compounds extracted from plants like Qinghaosu (artemisinin) (Li and Rieckmann, 1992) and it has stimulated the search for new plant derived drugs.
A part of our research program consists in the evaluation of the antiplasmodial activities of plants traditionally used in Indonesian regions to treat malaria, we have evaluated the antiplasmodial activity of some medicinal plants from South Kalimantan such as mahoni (Swientenia mahagoni Jack), brotowali (Tinospora tuberculata Beumee), mimba (Azadirachta indica A. Juss) and pasak bumi (Eurycoma longifolia Jack). Among aqueous extract of four plants tested, aqueous extract of E. longifolia showed strong antiplasmodial activity with an IC 50 value ranged from 1.07-5.64 µg mL −1 on chloroquine-sensitive (D-10) and-resistant (FCR-3) strains (Qamariah, 2002). In order to know the most potent extract of E. longifolia further study have been conducted. Three extracts of E. longifolia i.e., aqueous, methanol and chloroform extracts have been evaluated for their in vitro antiplasmodial activity and cytotoxicity (Mustofa and Qamariah, 2004). Among three extracts of E. longifolia tested, methanol extract exhibited a highest antiplasmodial activity with the IC 50 ranging from 0.6 to 1.9 µg mL −1 for the P. falciparum strains tested and its Cytotoxicity Index was higher (CI: 22.9-98.6) than chloroform extract (CI: 30.6-35.8) and lower than aqueous extract (CI: 132.6-142.6). The ethyl acetate soluble and insoluble fractions obtained from metahonic extract showed high antiplasmodial activity too (Mustofa and Sholikhah, 2007). Previous study of 5 isolates of methanol soluble fractions showed that isolate 4 showed a high in vitro antiplasmodial activity and high selectivity.
Phytochemical screening of E. longifolia extract showed that eurycumanone is the most active compounds contained in the plant's roots and potential as an antimalarial with IC 50 value of 48.1 ng mL −1 on chloroquine-resistant P. falciparum (W-2) and 47.7 ng mL −1 on the chloroquine sensitive P. falciparum (D-6) (Kardono et al., 1991). However, the stage of Plasmodium falciparum cycles which are sensitive to eurycomanone have not been investigated. Based on our previous achievements above mentions, continuation study has been planned in order to know which stage of P. falciparum sensitive for the activity. This study was intended to evaluate the stage specificity of eurycomanone isolated from E. longifolia root on P. falciparum cycles.

Materials
The E. longifolia roots were collected in Education Park Forest of Mulawarman University, South Kalimantan, Indonesia and were identified by comparison with authentic specimens. Eurycomanone was isolated in Department of Pharmacology and Therapy, parasite were obtained from the laboratory stock at the Department of Parasitology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta.

In vitro Stage Specificity Testing on Plasmodium falciparum
The FCR-3 strain of P. falciparum was used in this study. Parasites were cultured continuously according to Trager and Jensen (1976) with modifications described by Van Huysenn and Rieckmann (1993). The parasites were maintained in vitro in human red blood cells (O±), diluted to 3% hematocrit in RPMI 1640 medium supplemented with 25 mM Hepes and 30 mM NaHCO 3 and complement with 10% human O serum. Before used, parasite cultures were synchronized by D-sorbitol in order to obtained ring stage of P. falciparum as reported by Lambros and Vanderberg (1979). The stage specificity of eurycomanone was evaluated microscopically by observing the percentage of each stages of P. falciparum after 8, 16,24,32,40,48,56,64 and 72 h. incubation periods with 6 various concentration of eurycomanone compared with control without any compound.

Results
The results showed that giving eurycomanone with all concentrations i.e., 10, 20, 40, 60, 80 and 100 ng mL −1 on P. falciparum showed that the difference percentage of Plasmodium stage started at 8 h incubation period. At 8 h of incubation periods, untreated control Plasmodium showed 77.2% at the ring stage, whereas in Plasmodium which is given eurycomanone in concentration 10, 20, 40, 60, 80 and 100 ng mL −1 , there was a decrease in the percentage of ring r to 62.9; 57.7; 67.7; 69.8; 64.7 and 45.3% respectively. These results suggested that giving eurycomanone in all concentrations in this study can kill ring stage of Plasmodium.
At the 24-h incubation period, control Plasmodium showed that 7.23% had been in mature schizonts stage, whereas in Plasmodium which is given eurycomanone in concentartion 10, 20, 40, 60, 80 and 100 ng mL −1 respectively, showed only 1.11; 0; 1.85; 1.56; 0 and 1.39% only which were on mature schizont stage (Table 1). Plasmodium should have been in the mature schizont stage, but the eurycomane inhibited the growth of young schizont to mature schizont ( Fig. 1-6). Eurycomanone than can kill ring stage of Plasmodium, seems also inhibit the growth of young schizonts to mature schizonts. This condition caused the IC 50 value in 32, 40, 48, 56, 64 and 72 h of incubation time were declined (Table 2).

Discussion
The results showed that administration of eurycomanone in concentration 20, 40, 60, 80 and 100 ng mL −1 showed growth inhibition of young schizonts to mature scizont that it could be seen on a 24h incubation time. The IC 50 values in the 24-h incubation period could not be calculated. However since the incubation time of 32 h until the end of the study (72 h), the IC 50 were getting down (Table 2). Theoritically, at the 24th h, almost 50% Plasmodium grow into trophozoites and schizonts and at 32 h began to reinvasion into erythrocytes were not infected previously. At 48th h re-invasion has increased to about 40% (Srinivas and Puri, 2002). In this study, giving eurycumanone for 24h showed inhibit maturation of young schizont to mature schizont. If re-invasion started at 32 h, the observation could be performed at 40 h incubation period. At 40th h, control Plasmodium without any compound showed almost (95.78%) at ring stage. When compared with eurycomanone Plasmodium treated at concentration 10, 20, 40, 60, 80 and 100 ng mL −1 , percentage of ring stage of Plasmodium were lower (89.8; 94.54; 92.87; 82.26; 82.76 and 71.26% respectively). The greater the concentration of eurycomanone, the greater the inhibition.

Conclusion
Eurycomanone can kill ring stage of P. falciparum and may inhibit the development of young schizont to mature schizont in vitro. However, it needs further investigations for the mechanism.