34Pak Mus_pasak Bumi 2

AKTIVITAS ANTIPLASMODIUM in vitro dan in vitro FRAKSI YANG DIPEROLEH DARI EKSTRAK METANOL PASAK BUMI ( Eurycoma longifolia longifolia Jack) YANG SECARA TRADISIONAL DIGUNAKAN MENGOBATI MALARIA DI KALIMANTAN SELATAN

 In vitro AND in vivo ANTIPLASMODIAL ACTIVITY OF FRACTION ISOLATED FROM METHANOLIC EXTRACT OF PASAK BUMI ( Eurycoma longifolia Jack) TRADITIONALLY USED TO TREAT MALARIA IN S OUTH KALIMANTAN Mustofa & Eti Nurwening Sholikhah Department of Pharmacology and Toxicology/Center For Topical Medicine, Faculty of Medicine, Gadjah Mada University, Yogyakarta, ABSTRAK Akar pasak bumi ( Eurycoma longifolia Jack) secara tradisional digunakan untuk mengobati malaria di Kalimantasn Selatan. Ekstrak metanol pasak bumi terbukti aktif secara in vitro terhadap  P.  falciparum.  falciparum. Ekstrak metanol ini selanjutnya dilakukan fraksinasi dengan etil asetat. Penelitian ini dilakukan untuk mengkaji aktivitas antiplasmodium secara in vitro dan in vivo fraksi larut etil asetat dan fraksi tidak  larut etil asetat dari ekstrak metanol akar pasak bumi. Metodemikro radioaktif digunakan untuk mengkaji aktivitas antiplasmodium in vitro pada strain  P. falciparum resisten klorokuin, FCR-3. FCR-3. Pertumbuhan Pertumbuhan parasit parasit diperkirakan diperkirakan melalui ambilan [3H]hipoksantin oleh parasit setelah inkubasi 24 dan 72 jam. Uji baku 4 hari pada mencit yang diinfeksi  P. berghei digunakan untuk mengkaji aktivitas antiplasmodium secara in vivo. vivo. Apusan darah dibuat pada hari ke 5 setelah inokulasi dan pemeriksaan parasitemia dilakukan dengan mikroskop setelah pewarnaan Giemsa. Hasilnya ditunjukkan sebagai persen penghambatan pertumbuhan parasit setelah pemberian fraksi dibandingkan kontrol. Nilai ED50 selanjutnya ditetapkan dengan analisis probit. Hasil penelitian menunjukkan fraksi tidak larut etil asetat (IC 50, 0,314 ± 0,012 µg/mL setelah inkubasi 24 jam dan 0,061 ± 0,003 µg/mL setelah inkubasi 72 jam) lebih aktif terhadap  P. falciparum dibandingkan fraksi tidak larut etil asetat (IC 50, 1,499 ± 0,076 µg/mL setelah inkubasi 24 jam dan 0,388 ± 0,015 µg/mL setelah inkubasi 72 h jam). Fraksi tidak larut etil asetat (ED 50, 1,18 mg/Kg BB/hari) juga lebih aktif pada P. pada  P. berghei dibandingkan dengan fraksi larut etil asetat (ED 50, 6,40 mg/Kg BB/hari). Dari hasil  penelitian ini dapat disimpulkan fraksi tidak larut etil asetat lebih aktif baik pada uji in vitro maupun in vivo. vivo.  P. berghei – antiplasmodium –  E.  E. longifolia Jack - malaria Kata kunci : P. falciparum –  P. ABSTRACT Pasak bumi’s roots ( E. longifolia) longifolia) has been used traditionally to treat malaria in South Kalimantan. Preliminary study showed that methanol extract of pasak bumi roots was active in vitro against  P.  falciparum.  falciparum. This extract was subjected to a fractionation with ethyl acetate. This study was conducted to evaluate the in vitro and in vivo antiplasmodial activity of ethyl acetate soluble and ethyl acetate insoluble fractions obtained from methanolic extract of the pasak bumi roots. A radioactive micromethod was used to the in vitro antiplasmodial activity on a FCR-3 chloroquine-resistant strain of  P. of  P. falciparum. falciparum. The parasite growth was estimated by [ 3H]-hipoxanthine incorporation after 24 and 72 hours incubation. A standard 4-day test on P. on  P. berghei infected mice was used to evaluate the in vivo antiplasmodial activity of the fractions. Blood film was prepared on the 5th day after   parasite inoculation and Giemsa stained. The results were expressed as percentage inhibition of parasite growth as compared to the control groups. The ED50 values were determined by probit analysis. The results showed that the ethyl acetate insoluble fraction (IC 50, 0,314 ± 0,012 µg/mL after 24 h incubation and 0,061 ± 0,003 µg/mL after 72 h incubation) was more active against  P. falciparum than ethyl acetate fraction (IC50, 1,499 ± 0,076 µg/mL after 24 h incubation and 0,388 ± 0,01 µg/mL after 72 h incubation). The ethyl acetate insoluble fraction (ED50 = 1.18 mg/Kg BW/d) was also more active against  P. berghei in infected mice than the ethyl acetate fraction (ED 50 = 6.40 mg/Kg BW). It can be concluded that the ethyl acetate insoluble fraction was more active than the ethyl acetate soluble fraction (in (in vitro and in vivo). vivo).  P. berghei – antiplasmodial –  E.  E. longifolia longifolia Jack - malaria Key words : P. falciparum –  P.

INTRODUCTION

Malaria is still a health problem in tropical and sub-tropical countries placing at risk  some 40% of the world’s population. More than 100 million clinical cases of the disease are thought to occur annually resulting at least in 12 million deaths (WHO, 1997; 1998). The increasing resistance of  Plasmodium falciparum strains to currently available antimalarial has initiated numerous studies aimed at identifying new antimalarial agents. One of the strategies in search for new antimalarial compounds is a research of active plant constituents. Medicinal  plants have been used traditionally to treat of  malaria in endemic area in the world where malaria prevails. Significant success was achieved with the new compounds extracted from plants such as Qinghaosu (artemisinin) (Li and Reickmann, 1992). This success has stimulated the search for new plant derived drugs. A part of our research program consists in the search of antimalarial compound from  plants traditionally to treat malaria in Indonesia, we have evaluated the antiplasmodial activity of  some medicinal plants from South Kalimantan viz. mahoni (Swientenia mahagoni Jack),  penawar sampai (Tinospora tuberculata Beumee.), mimba ( Azadirachta indica A. Juss.), and pasak bumi ( Eurycoma longifolia Jack). Among aqueous extract of four tested plants, aqueous extract of  E. longifolia Jack showed strong antiplasmodial activity with an IC 50 value ranging from 1.07 – 5.64 µg/mL on chloroquinesensitive (D-10) and –resistant (FCR-3) strains (Mustofa & Sholikhah, 2002). In order to know the most potent extract of   E. longifolia Jack   , further study has been conducted. Three extracts of  E. longifolia Jack i.e. aqueous, methanol, and chloroform extracts have been evaluated for  their  in vitro antiplasmodial activity and cytotoxicity (Mustofa & Qamariah, 2004). Among the three extracts of tested  E. longifolia Jack, methanol extract exhibited the highest antiplasmodial activity with the IC 50 ranging from 0.6 to 1.9 µg/mL for the tested  P.  falciparum strains tested and its Cytotoxicity Index was higher (CI : 22.9 – 98.6) than

chloroform extract (CI : 30.6-35.8) but was lower than aqueous extract (CI : 132.6-142.6). On the basis of our previous achievements mentions above, further study has been planed in order to know which fractions of methanol extract of  E. longifolia Jack roots showing the most potent antiplasmodial activity. In this study methanol extract of  E. longifolia Jack was fractioned with ethyl acetate and the fractions obtained were evaluated for their  in vitro and in vivo antiplasmodial activity. MATERIALS AND METHODS Materials The  E. longifolia Jack roots were collected in Education Park Forest of  Lambung Mangkurat University, South Kalimantan, Indonesia and were identified by comparison with authentic specimens at Laboratory of Pharmacy Biology, Faculty of  Pharmacy, Gadjah Mada University (GMU), Yogyakarta. A voucher specimen was deposited at the Department of Pharmacology and Toxicology, GMU. Parasites were obtained from the laboratory stock at the Life Science Laboratory collection, GMU. Plant fractions preparation The  E. longifolia Jack root was air  dried and powdered. The extracts were then  prepared by maceration of the powder (1,5 kg) with 7 L methanol at room temperature for 24 hours. The process was repeated three times. The filtrates were then combined and evaporated under vacuum to dryness. The methanol extract was then made into fraction with ethyl acetate. The filtrates of ethyl acetate were concentrated by a rotary evaporator to obtain ethyl acetate soluble fraction whereas the residues were evaporated to obtain ethyl acetate insoluble fraction. The fractions were then refreshed at 4 Ο C until the analysis was conducted. Parasite strain and in vitro culture

A strain of   P. falciparum, FCR-3 chloroquine resistant strain was used in the study. Parasites were cultured continuously

according to Trager & Jensen (1976), with modifications described by Van Huyssen & Rieckmann (1993). The parasites were maintained in vitro in human red blood cells (O ±), diluted to 1% hematocrit in RPMI 1640 medium, supplemented with 7.68 mM Hepes and 23.78 mM NaHCO3, and complemented with 10% human O serum. Before using, parasite cultures were synchronized by a D-sorbitol lysis in order  to obtain ring stage of  P. falciparum as reported  by Lambros & Vanderberg (1979).  In vitro antiplasmidial activity testing The antiplasmodial activity of   E. longifolia Jack fractions was evaluated by a radioactive method described by Desjardins et  al ., (1979). Fractions were tested in triplicate in 96-well culture plates. Parasites mostly at ring stages were suspended in RPMI 1640 medium containing 10 mM NaHCO3 to 1% parasitemia and 2 % hematocrit and distributed into wells of  a 96-well micro plate (100 µl per well).  Eurycoma longifolia Jack roots fractions at various concentrations in RPMI 1640 medium were added into the wells. The micro plate containing parasite culture and  E. longifolia Jack  roots fractions was then incubated in 37 0C candle jar incubator for two-time interval, 24-h and 72-h. The control of parasite cultures freed from any fractions was referred to as 100% growth. Parasite growth was estimated by [ 3H]hypoxanthine incorporation. After 18-h or 60-h incubation, 50 µl of culture medium containing 0.25 µCi [3H]-hypoxanthine was added into each well and the parasites were incubated for  the further 6-h or 12-h. In the end of the incubation period, the content of each well was harvested on glass fiber filter papers with a Cell Harvester and dried for 2-h at 60 0C. The radioactivity of each well was measured by a liquid scintillation counter. The net incorporation of radioactivity was obtained after  subtraction of the non-specific incorporation measured in uninfected erythrocytes, and expressed as a percentage of the incorporation  by the positive controls. The IC 50 values, concentration required to inhibit parasite growth  by 50%, were determined by linear interpolation

from the growth inhibition curves generated for each extract-parasite combination.

 In vivo antiplasmodial testing The in vivo antiplasmodial activity of the extracts was determined by the classic 4-day suppressive test against  P. berghei, NK 65 strain, in mice (Peters et al., 1975). Thirty Swiss male mice weighing 20 ± 2 g were inoculated with 1 x 10 6 infected red blood cells intraperitoneally on day 0. At 2 h after  inoculation, mice were injected intraperitoneally with 0.1 mL of extracts tested in water solution at different concentration, given doses ranging from 50 to 400 mg/kg of   body weight/day. Eight animals were tested at each dose level. The injection of tested extracts was repeated daily for 3 consecutive days from day 0 of parasite injection. The control group treated with distilled water was used in all extracts testing. On the 5 th day after   parasite inoculation, blood films were taken from the tail blood and the level of parasitemia determined on Giemsa stained smears by counting 2000 erythrocytes. Parasite growth inhibition by extracts was determined by comparison with the level of parasitemia in control group. For the 4-day suppressive method, ED50 values were determined by  probit analysis. RESULTS AND DISCUSSION  Eurycoma longifolia, Jack is a small tree from the family Simarobaceae and it can be found in the jungle throughout Malaysia, Indonchina, and Indonesia especially in Sumatra and Kalimantan. Eurycoma longifolia Jack is often used by traditional healers to treat various diseases. A decoction of the root  bark is drunk to treat diarrhea, fever, glandular  swelling, bleeding, dropsy, persistent cough and hypertension. It also relieves pain in the  bones. Besides, it is used as an aphrodisiac and tonic. The pounded bark is applied to treat wounds, ulcers, syphilitic sores, and headache (Chan et al., 1991; Bedir  et al ., 2003). Pharmacological study of this plant showed that the  E. longifolia Jack exhibited various  biological activities such as antiulcer,

cytotoxic, antipyretic, and antimalaria (Ang and Sim, 1997). In South Kalimantan, Indonesia,  E. longifolia Jack plays an important role in the treatment of various diseases such as infections, diarrhea and fever in malaria. Our previous study showed that among the three extracts of  tested pasak bumi root viz. aqueous, chloroform, and methanol extracts, methanol extract exhibited the highest antiplasmodial activity

(Mustofa & Qamariah, 2004). In this study, we evaluated in vitro and in vivo antiplasmodial activity of ethyl acetate soluble and ethyl acetate insoluble fractions of the methanol extract. The effect of these extracts on growth inhibition of  P. falciparum (FCR-3 strain) were shown in Figure 1 and Table 1.

100

80    )    %    (

  n 60   o    i    t    i    b    i    h   n    i    h    t 40   w   o   r    G

ethyl acetate soluble 24-h ethyl acetate soluble 72-h

20

ethyl acetate insoluble 24-h ethyl acetate insoluble 72-h

0 0

1

2

3

4

5

Concentration (ug/mL)

Figure 1. Growth inhibition of  P. falciparum (FCR-3) treated by ethyl acetate soluble and ethyl acetate insoluble fractions of pasak bumi ( E. longifolia) roots.

Table 1. Growth inhibition of  P. falciparum (FCR-3) treated by ethyl acetate soluble and ethyl acetate insoluble fractions of pasak bumi ( E. longifolia Jack) roots. Concentration (µg/mL)

ethyl acetate soluble fraction (%± SEM)

ethyl acetate insoluble fraction (%± SEM)

24-h

72-h

24-h

72-h

0,1

17.3 ± 1.5

56.7 ± 2.2

0±0

24.8 ± 6.6

0,5

52.1 ± 1.6

93.6 ± 0.7

12.8 ± 2.1

53.0 ± 7.5

1,0

80.6 ± 1.1

96.5 ± 0.7

32.8 ± 3.1

79.1 ± 7.1

2,0

89.1 ± 0.8

90.0 ± 0.6

67.3 ± 3.5

97.2 ± 1.2

5,0

91.8 ± 0.7

97.1± .3

86.0 ± 2.1

96.1 ± 1.2

IC50 (µg/mL) SEM

0,314 0,012

0,061 0,003

1,499 0,076

0,388 0,015

As shown in Table 1, the ethyl acetate soluble and ethyl acetate insoluble fractions exhibited a higher  in vitro antiplasmodial activity with the IC 50 values ranged from 0,061 to 1,499 µg/mL, depending on incubation time and kind of fraction. In comparison with the time of incubation, significant differences were observed between the IC 50 values after 24-h and 72-h incubation time. The both fractions were more active after 72-h contact between the  parasite and the fraction than after 24-h contact indicating the existence cumulative effect. The ethyl acetate insoluble fraction of pasak bumi showed higher  in vitro antiplasmodial activity (IC50, 0,061 to 0,314 µg/mL) than ethyl acetate soluble fraction (IC 50, 0,388 to 1,499 µg/mL) indicating that more active compounds could be isolated with a semi polar or a polar solvent. These results of  in vitro test were considered sufficient to be evaluated for the in vivo antiplasmodial activity. For this purpose, a 4-day suppressive test was performed on male Swiss mice using  P. berghei. The results of the in vivo test were summarized in Table 2. These obtained results were consistent with values obtained from in vitro test, in which the ethyl

acetate insoluble fraction was more active (ED50, 1.18 mg/kg. BW/d) than ethyl soluble fraction (ED50, 6.40 mg/kg. BW/d). However, the in vivo antiplasmodial activity of the both fractions was lower than chloroquine as  positive control (ED50, 0.67 mg/kg. BW/d). The in vivo antiplasmodial activity of an extract or a fraction can be classified as moderate, good and very good activity if the extract displayed a percent parasite growth inhibition is equal or greater than 50% at the dose 500, 250 and 100 mg/kg BW/d, respectively (Munoz et al., 2000). Based on the classification, the both ethyl acetate soluble and ethyl acetate insoluble fractions of   E. longifolia Jack exhibited very good antiplasmodial activity. Phytochemical studies on  E. longifolia Jack  showed that it possesses a series of triterpenes, squalene derivatives, lignans, alkaloids, and quassinoids (Chan et al., 1991; Bedir  et al ., 2003). The quassinoids are one of the major   bioactive groups in this plant and show a large spectrum of biological activities, which include antitumor, antiviral, antiamoebic, antiamoebic, and antiplasmodial (Polonsky, 1985; Wright and Philipson, 1990).

Table 2.  In vivo antiplasmodial activity (ED 50 in mg/kg.BW/d ± SEM) of ethyl acetate soluble and ethyl acetate insoluble fractions of pasak bumi ( E. longifolia Jack) roots on P. berghei infected mice.

Fraction Ethyl acetate soluble Estimated ED50 = 6.40 mg/kg. BW/d

Ethyl acetate insoluble Estimated ED50 = 1.18 mg/kg.BW/d

Chloroquine Estimated ED50 = 0.67 mg/kg.BW/d

Controla

Dose tested (mg/kg.BW/d)

Parasitemia

Growth inhibition

Mortality

(% ± SEM )

(% ± SEM)

(n/N ) b

1.25 2.50 5.00 10.00 20.00 1.25 2.50 5.00 10.00 20.00 1.00 2.00 4.00 8.00 16.00 -

67.11 ± 3.70 68.94 ± 7.38 67.95 ± 6.05 24.45 ± 2.87 15.04 ± 2.41 24.39 ± 2.82 17.13 ± 3.03 1.55 ± 0.49 0.85 ± 0.27 0.02 ± 0.00 25.33 ± 4.36 6.42 ± 2.25 0.40 ± 0.09 0.01 ± 0.0 0.0 ± 0.0 66.68 ± 1.79

0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 63.33 ± 5.50 85.90 ± 5.35 63.42 ± 5.57 74.30 ± 4.24 97.50 ± 0.67 98.50 ± 0.23 99.75 ± 0.07 62.00 ± 6.11 89.33 ± 3.63 99.25 ± 0.15 99.83 ± 0.04 100.00 ± 0.00 0.0 ± 0.0

0/6 0/6 0/6 3/6 1/6 0/6 1/6 1/6 3/6 3/6 1/6 0/6 0/6 0/6 1/6 3/6

Ang et al. (1995) have isolated some quassinoids from  E. longifolia Jack  traditionally used to treat febrifuge and malaria in Malaysia viz. 13 β ,18dihydroeurycomanol, eurycomanol-2- β -18D-glucopyranoside, eurycomanol and eurycomanone and reported their  in vitro antiplasmpodial on the Malaysian  P.  falciparum isolates. Jiwajinda et al. (2001) have isolated new derivatives quassinoids from E. longifolia Jack such as longilactone, dehydrolongilactone, 11dehydroklaineanone, 15βhydroxyklaineanone, 14,15βdihydroxyklaineanone and 15 β-O-acetyl-14hydroxyklaineanone during the course of the search for plant growth inhibitors occurring in the medicinal plants of Thailand. Moreover, these quassinoids have been  proven to have antiplasmodial activity on the W2 chloroquine resistant strain of   P.  falciparum  by Jiwajinda et al. (2002). Thus, it should be noted that the different derivatives of quassinoids could be found from regions. Finally, the new type of  quassinoids with a potential antiplasmodial activity could be obtained from  E. longifolia Jack from South Kalimantan. CONCLUSION In conclusion, the present study showed that the ethyl acetate insoluble fraction isolated from methanol extract of  E. longifolia Jack displayed better  antiplasmodial activity (in vitro and in vivo), than that of ethyl acetate soluble fraction. Further study including the purification of the  bioactive compounds and search for possible its mechanism of action will be focused on the ethyl acetate insoluble fraction.

AKNOWLEDGMENTS

This work is supported by Public Fund, Faculty of Medicine, Gadjah Mada University at 2002. The authors wish to thank  Mr. Sukiran and Mrs. Rumbiwati for the excellent technical assistance.

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