In Vivo Antiplasmodial of the Most Active Fraction and Its Compound of Kapur Leaves (Harmsiopanax Aculeatus Harms) Extract Against Plasmodium Berghei

TMJ • Vol. 01 • No.02 • 131-140

In vivo Antiplasmodial of the Most Active Fraction and Its Compound of Kapur Leaves (Harmsiopanax aculeatus Harms) Extract Against Plasmodium berghei

Rachel Turalely1, Ratna Asmah Susidarti2, Mahardika Agus Wijayanti3 1Department of Mathematics and Education Science, Faculty of Teaching and Education science, Universitas Pattimura. 2Departement of Chemistry Pharmacy, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta; 3Departement of Parasitology, Faculty of Medicine, Universitas Gadjah Mada,Yogyakarta, Indonesia

*Corresponding author: [email protected]

ABSTRACT Introduction : The rising of Plasmodium resistance towards chloroquine and other antimalarial drugs have encouraged to discover and develop new drugs mainly derived from natural products. Harmsiopanax aculeatus (kapur plant) has traditionally used by people of in Maluku Province to treat malaria. Objectives: The aims of this study were to identify antiplasmodial activity and its chemical constituents of the most active fraction of kapur leaves. Methods: The dried powder of Kapur leaves (1.3 kg) were extracted successively by maceration with n-hexane, ethyl acetate and methanol. After removal the solvents the hexane 15.6 g (1.2%), ethyl acetate 53.3 g (4.1%) and methanol 61.1 g (4.7%) extracts were obtained. Those extracts were assayed for their in vivo antiplasmodial activities by using 4-days suppressive test in Swiss mice infected with Plasmodium berghei, HPIA and identified the compound by GC-MS.

Results: The ED50 of hexane, ethyl acetate and methanol extracts were 467.58, 2074.02 and 16.16 mg/ kgBW, respectively. Fractionation of the methanol extract gave 18 combined fractions (FG1 – FG18). FG8 was the most active fraction with the IC50 HPIA of 18.22 µg/ml. Phytochemical test of this fraction using spray reagent showed the existence of essential oils, triterpenoids, and phenolic compounds. Separation of FG8 using pressed chromatography gave 19 combined fractions (FG8.1-FG8.19). The fraction containing intense blue fluorescent spot (FG8.5) was further separated by PLC fourthly eluted with chloroform. Seven major components with the percentage of compotition more than 3.11% were identified as eugenol (tr = 12.692;

18.22%), isoprophyl myristate (tr = 16.333; 3.99%); bis(2-methylpropyl) phtalat (tr = 16.939; 7.15%); methyl palmitic (tr = 17.442; 3.11%); palmitic acid (tr = 17.883; 25.72%); butyl 2-methylpropyl phtalat (tr = 17.957;

9.37%) and bis(2-ethylhexyl) phtalat (tr = 23.258; 23%). Conclusion: Methanol extract of H. aculeatus was the most potential in vivo antiplasmodial activity. Combined fraction 8 which contain 7 compounds was the most active fraction.

Key words: Harmsiopanax aculeatus Harms, in vivo antiplasmodial, HPIA, PLC, GC-MS

INTISARI Pendahuluan: Adanya resistensi Plasmodium terhadap klorokuin dan obat antimalaria lainnya telah mendorong dilakukannya penelitian untuk mencari dan mengembangkan obat baru terutama yang berasal dari bahan alam. Daun tanaman kapur (Harmsiopanax aculeatus Harms) telah digunakan secara tradisional oleh masyarakat Maluku untuk mengobati malaria. Tujuan: Serbuk kering daun kapur sebanyak 1,3 kg diekstraksi secara maserasi bertingkat menggunakan n-heksan, etil asetat dan metanol. Setelah pelarut diuapkan diperoleh ekstrak heksan sebesar 15,6 g (1,2%), ekstrak etil asetat 53,3 (4,1%) dan ekstrak metanol 61,1 (4,7%). Ekstrak diuji aktivitas antiplasmodiumnya secara in vivo menggunakan metode 4 days suppressive test pada mencit Swiss yang diinfeksi Plasmodium berghei, HPIA dan identifikasi senyawa dengan GC-MS

131 Rachel Turalely et.al, In vivo Antiplasmodial of the Most Active Fraction and Its Compound of Kapur Leaves (Harmsiopanax aculeatus Harms) Extract Against Plasmodium berghei

Hasil: ED50 ekstrak heksan, etil asetat dan metanol berturut turut adalah 467,58; 2074,02 dan 16,16 mg/ kgBB. Fraksinasi ekstrak metanol dilakukan dengan VLC menggunakan silika gel yang dielusi dengan meningkatkan polaritas pelarut, menghasilkan 18 fraksi gabungan (FG). 18 FG ini diuji HPIA secara in vitro.

FG8 adalah fraksi paling aktif dengan IC50 HPIA 18,22 µg/ml. Uji fitokimia terhadap fraksi ini menggunakan pereaksi semprot memperlihatkan keberadaan minyak atsiri, triterpenoid dan senyawa fenolik. Pemisahan

FG8 menggunakan kromatografi tekan menghasilkan 19 fraksi gabungan8.1 (FG – FG8.19). Fraksi yang mengandung spot berfluoresensi biru (FG8.5) dipisahkan menggunakan PLC 4 kali elusi dengan kloroform. Pita berfluoresensi biru dikerok dan diekstraksi dengan kloroform. Isolat kemudian dianalisis menggunakan GC-MS. Tujuh komponen utama dengan komposisi persentase lebih dari 3,11% diidentifikasi sebagai eugenol (tr = 12,692; 18,22%), isopropil miristat (tr = 16,333; 3,99%); bis(2-metilpropil) ftalat (tr = 16,942;

7,15%); metil palmitat (tr = 17,442; 3,11%); asam palmitat (tr = 17,883; 25,72%); butil 2-metilpropil ftalat (tr =

17.957; 9.37%) dan bis(2-etilheksil) ftalat (tr = 23,258; 20,29%). Simpulan: Ekstrak metanol H. aculeatus mempunyai aktivitas antiplasmodium in vivo yang paling potensial. Fraksi gabungan 8 merupakan fraksi yang paling aktif mengandung 7 komponen utama.

Kata kunci: Harmsiopanax aculeatus Harms, antiplasmodium in vivo, HPIA, PLC, GC-MS

Introduction is the emergence of Plasmodium resistance to antimalarial drugs, particularly chloroquine, Malaria is an infectious disease caused by known previously as first-line drugs. Perez et al4. parasites of the genus Plasmodium1. Based states that the global spread of malaria parasites on data from 2009 and WHO’s Global Malaria resistant to several drugs that are available is a Action Plan, 3.3 billion people (half the human major health problem that requires effort to find population) live in areas with malaria transmission new antimalarial drugs. One attempt was made and thirty-five countries (30 in sub-Saharan through the study of medicinal plants used by Africa and 5 in Asia) has a high mortality caused traditional communities to treat malaria. Plant by malaria by 98%. In 2008, malaria is estimated of Harmsiopanax aculeatus (Bl. Ex DC.) Harms at 190-311000000 clinical symptoms with is a traditional medicinal plants, which is widely mortality rates as much as 708,000-1,003,000 used by people in Maluku to treat malaria. people2. The spread of malaria is widespread in Maluku people call it “Kapur “. The aplication many developing countries including Indonesia. is that clean young lime leaves pressed, filtered Indonesia is one country that is still considered with a clean cloth and then dropped into the at risk of malaria and frequent outbreaks eyes of patients with malaria one drop / day (epidemics). Various measures of prevention for three consecutive days. Kapur plant are and treatment to combat malaria have been classified into Araliaceae family and based on conducted such as vector control, use of a literature search has not been carried out antimalarial drugs, the use of anti-mosquito bed phytochemical and pharmacological studies nets and other means have given positive results on this plant. This research was conducted to in limiting the spread of this disease, but the obtain the most active antiplasmodial fraction eradication of malaria is still far from perfect, of kapur leaf and identity of the compounds even some areas the situation becomes more using GC-MS. This study uses Bioassay Guided worse with the emergence of parasite strains fractionation. Kapur leaves was extracted using resistant to more than one type of anti-malarials3. n-hexane, ethyl acetate and methanol. Hexane, One cause of failure of eradication of malaria ethyl acetate and methanol extracts of kapur

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leaves were then tested for antiplasmodial mice were divided over 18 groups. The first 5 was sown on the stationary phase until blended nm. Absorbance values obtained are plotted to activity in vivo against Plasmodium berghei. groups received 25, 50, 100, 200 and 400 mg/ and the powder surface was closed again with a linear regression equation of standard curve The most active extract was then separated by kgBW/d hexane extract, respectively. The second silica gel 40 Merck (37-50 mesh). Performed so that it can be determined the concentration chromatography and fractions obtained tested 5 groups received 12.5, 25, 50, 100 and 200 mg/ by gradient elution starting with hexane- ŽĨ ɴͲŚĞŵĂƚŝŶ ŽĨ ƚŚĞ ƐĂŵƉůĞƐ͘ sĂůƵĞƐ ŽĨ ŚĂĞŵ for haem polymerization inhibition activity. The kgBW/d ethyl acetate extract. The third 7 groups chloroform, followed by chloroform-methanol polymerization inhibition IC50 were expressed in most active fraction is further separated and the received 1.25, 2.5, 5, 10, 20; 40 and 80 mg/kg and finally methanol with various comparisons. levels that could inhibit the polymerization hem compounds were analyzed by GC-MS. /d methanol extract, respectively. The other Fractions obtained, evaporated the solvent and up to 50%. group without compound was negative control. TLC profile of his views. Fractions with similar Analysis of Group Compounds in The Most

Each group consisted of 5 male and 5 female TLC profiles were combined. A number of 18 Active Fraction (FG8) of Methanol Extract. FG8 Materials and Methods mice. Each dose of the extract was prepared by combined fractions were obtained and tested solution in chloroform spoted on 7 plates thin dissolved in DMSO at 8%. Each dose of extract for haem polymerization inhibition activity. layer of silica gel 60 F254 (1.05735) measuring 1x9 Plant Extract. Harmsiopanax aculeatus was was administered once daily for 4 consecutive Haem Polymerization Inhibition Activity. cm and eluted using eluent system chloroform obtained from the Amahai village and district of days, beginning on the day of infection, starting Haem polymerization inhibition test was ethyl acetate (9:1). TLC plates were then sprayed

Central Maluku, determined at the Laboratory of two hours after inoculation until third day (H3). carried out on each combined fraction by using with Dragendorff reagent, FeCl3, Liebermann- Plant Taxonomy Faculty of Biology, Universitas Parasitaemia was calculated using thin blood Bassilico et al. method6 with modification. Put Burchard, KOH and Anisaldehid sulfate. Change Gadjah Mada. H. aculeatus leaves were dried smears which were stained by Giemsa and a 100 µL solution of 1 mM hematin in 0.2 M the color of the spots seen in visible light and and 1.3 kg of powder was made. The dried examined under a light microscope. The level of NaOH and 50 µL material tested into eppendorf UV 365 nm and compared with the standard TLC powder of Kapur leaves (1.3 kg) were extracted parasitaemia was determined the day following tube, then added 50 µL of glacial acetic acid plates. successively by maceration with n-hexane, ethyl the last treatment (fourth day). The ED50, which (pH 2.6). Various concentration of materials Fractionation of combined fractions 8 acetate and methanol. is the dose leading to 50% parasite growth tested were used : 25; 50; 100; 200; 400 and methanol extract of H. aculeatus. Fractionation

Materials to extraction, fractionation, inhibition compared to growth in the control, 800 µg/ml for the combined fractions 1-7 and of FG8 methanol extract of kapur leaves isolation and identification compound. Material was evaluated from a plot of activity (expressed 9-18, while a series of concentration 6.25, 12.5, performed using column chromatography press of extraction are n-hexane, ethyl acetate, as a percentage of the activity in the control) 25; 50; 100; 200; 400 and 800 µg/ml were used with 1cm column diameter. Active fraction that methanol technical level. Materials for insulation versus the log dose. for combined fraction of 8. Each concentration was used as much as 0.1323 grams dissolved is a paper filter and lime leaves young tubers, Fractionation of Methanol Extract H. was made triplicate. Aquadest and chloroquine in chloroform and then impregnated with chloroform, ethyl acetate, methanol analysis of aculeatus. The most potential antiplasmodial were used for negative and positive control. 0.5292 gram of silica gel Merck 40 PF254 (37- degree; technical ethanol, silica gel GF40 (35- activity was methanol extract. The extract then Chloroquine concentrations were 25; 50; 100; 50 mesh) . Chloroform was evaporated so that 37 mesh), and silica gel 60 GF pore size of 230- fractionated using vacuum liquid cromatography 200; 400 and 800 µg/ml. Eppendorff which the obtained samples powder is ready for the 400 mesh (E. merck production ), silica gel 60H (VLC). The amount of 10 grams of methanol already contains the solution was incubated at fractionated. The column was prepared by the (1105932), silica gel GF254, filter paper, aquades, extract was dissolved in methanol and then 370C for 24 hours. After incubation, eppendorff solvent n-hexane and then silica slurry made silica gel F254 plates (1.05735) and plate PLC. impregnated with silica gel 40 Merck (37- was centrifuged at 8000 rpm for 10 minutes and of silica gel 60 PF254 Merck (230-400 mesh) Materials to haem polymerization inhibition 50 mesh). Methanol solvent was evaporated then washed three times with DMSO. Precipitate filled slowly inserted into the column and left test. Hematin(Sigma), 0.1 M NaOH, 0.2 M NaOH, until the mixture becomes dry. Column of haematin crystal was dissolved with 200 mL overnight. Furthermore, the samples powder 100% glacial acetic acid, DMSO, aquades, chromatography provided the solvent n-hexane of NaOH 0.1 M. A total of 100 mL solution was are inserted at the top and coated with silica Chloroquine diphosphat. ± ¾ stationary phase column and then powdered transferred to microplate 96 wells and measure gel Merck 40 PF254 (35-70 mesh). Fractionation In vivo antiplasmodial activity. silica gel 60H which had been mixed with the ƚŚĞĂďƐŽƌďĂŶĐĞǀĂůƵĞƐďǇ>/^ZĞĂĚĞƌĂƚʄϰϬϱ performed gradient using a ratio of chloroform- Antiplasmodial activity was evaluated by 4-day solvent n-hexane in the form of silica slurry, was suppressive test method5. Male and female Swiss added to the column a little while vaccumed The percentage inhibition of haem polymerization calculated by the formula: mice (20-25g) were inoculated intraperitoneally up to ± 15 cm high column. Columns that have ΀ȲͲŚĞŵĂƚŝŶ΁negative controlͲ΀ɴͲŚĞŵĂƚŝŶ΁materials testing with 107 P. berghei-infected erythrocytes, been packed, left overnight. The powder sample % Inhibition = x 100% resuspended in RPMI 1640 medium on day 0 was sown on the stationary phase until blended ΀ȲͲŚĞŵĂƚŝŶ΁ negative control (H0) to a volume of 0.2 ml. One hundred eighty and the powder surface was closed again with

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silicawas sown gel 40on the Merck stationary (37-50 phase mesh). until Performed blended nm.so that Absorbance it can be valuesdetermined obtained the areconcentration plotted to byand gradientthe powder elution surface starting was closed with again hexane- with aof linear β-hematin regression of the equation samples. of Values standard of haemcurve chloroform,silica gel 40 followed Merck (37-50 by chloroform-methanol mesh). Performed sopolymerization that it can be inhibition determined IC50 the were concentration expressed andby gradientfinally methanol elution with starting various with comparisons. hexane- ŽĨin levelsɴͲŚĞŵĂƚŝŶ that ŽĨ could ƚŚĞ inhibitƐĂŵƉůĞƐ͘ the sĂůƵĞƐ polymerization ŽĨ ŚĂĞŵ

Fractionschloroform, obtained, followed evaporated by chloroform-methanol the solvent and polymerizationhem up to 50%. inhibition IC50 were expressed in TLCand finally profile methanol of his views. with Fractionsvarious comparisons. with similar levelsAnalysis that could of Group inhibit Compounds the polymerization in The Most hem

TLCFractions profiles obtained, were combined.evaporated A the number solvent of and 18 upActive to 50%. Fraction (FG8) of Methanol Extract. FG8 combinedTLC profile fractions of his views. were Fractions obtained with and similar tested solutionAnalysis in chloroform of Group Compounds spoted on 7in plates The Most thin forTLC haem profiles polymerization were combined. inhibition A number activity. of 18 Activelayer of Fraction silica gel 60(FG F2548) of (1.05735) Methanol measuring Extract. 1x9FG8 combinedHaem fractionsPolymerization were obtained Inhibition and Activity tested. solutioncm and eluted in chloroform using eluent spoted system on 7 chloroform plates thin Haemfor haem polymerization polymerization inhibition inhibition activity. test was layerethyl ofacetate silica gel (9:1). 60 F254TLC plates (1.05735) were measuring then sprayed 1x9 carriedHaem out Polymerizationon each combined Inhibition fraction by Activity using. cmwith and Dragendorff eluted using reagent, eluent FeCl3,system Liebermann- chloroform BassilicoHaem polymerizationet al. method 6 with inhibition modification. test was Put ethylBurchard, acetate KOH (9:1). and TLC Anisaldehid plates were sulfate. then sprayedChange carrieda 100 µLout solution on each ofcombined 1 mM hematin fraction inby 0.2 using M withthe color Dragendorff of the spots reagent, seen FeCl3, in visible Liebermann- light and BassilicoNaOH and et 50 al. µL method material6 with tested modification. into eppendorf Put Burchard,UV 365 nm KOH and andcompared Anisaldehid with the sulfate. standard Change TLC atube, 100 then µL solution added 50 of 1µL mM of hematin glacial acetic in 0.2 acid M theplates. color of the spots seen in visible light and NaOH(pH 2.6). and Various50 µL material concentration tested into of eppendorf materials UV 365Fractionation nm and compared of combined with the standard fractions TLC 8 tube,tested then were added used 50: 25; µL 50; of 100; glacial 200; acetic 400 acidand plates.methanol extract of H. aculeatus. Fractionation

(pH800 µg/ml 2.6). Various for the combinedconcentration fractions of materials 1-7 and of Fractionation FG8 methanol of extract combined of kapur fractions leaves 8 tested9-18, while were a seriesused :of 25; concentration 50; 100; 200; 6.25, 400 12.5, and methanolperformed extract using column of H. aculeatus chromatography. Fractionation press

80025; 50; µg/ml 100; for200; the 400 combined and 800 fractionsµg/ml were 1-7 used and ofwith FG 1cm8 methanolcolumn diameter. extract Active of kapur fraction leaves that 9-18,for combined while a series fraction of concentrationof 8. Each concentration 6.25, 12.5, performedwas used as using much column as 0.1323 chromatography grams dissolved press in 25;was 50; made 100; triplicate. 200; 400 Aquadest and 800 µg/mland chloroquine were used withchloroform 1cm column and then diameter. impregnated Active fraction with 0.5292 that forwere combined used for fraction negative of 8. and Each positive concentration control. wasgram of used silica as gel much Merck as 0.132340 PF254 grams (37-50 dissolved mesh) . wasChloroquine made triplicate. concentrations Aquadest were and 25;chloroquine 50; 100; inChloroform chloroform was evaporated and then so impregnated that the obtained with were200; 400used andfor negative 800 µg/ml. and Eppendorff positive control. which 0.5292samples gram powder of silica is ready gel Merck for the 40 fractionated. PF254 (37- Chloroquinealready contains concentrations the solution were was incubated 25; 50; 100; at 50The mesh) column . Chloroform was prepared was evaporated by the solventso that 200;370C for 400 24 and hours. 800 After µg/ml. incubation, Eppendorff eppendorff which then-hexane obtained and samples then silica powder slurry is made ready of for silica the alreadywas centrifuged contains at the 8000 solution rpm for was 10 incubatedminutes and at fractionated.gel 60 PF254 MerckThe column (230-400 was mesh) prepared filled byslowly the 37then0C washedfor 24 hours. three Aftertimes incubation,with DMSO. eppendorff Precipitate solventinserted n into-hexane the and column then and silica left slurry overnight. made wasof haematin centrifuged crystal at 8000 was rpmdissolved for 10 withminutes 200 andmL Furthermore,of silica gel 60the PF254 samples Merck powder (230-400 are inserted mesh) thenof NaOH washed 0.1 threeM. A totaltimes of with 100 DMSO. mL solution Precipitate was atfilled the slowly top and inserted coated into with the silica column gel Merck and left40 oftransferred haematin to crystal microplate was dissolved 96 wells andwith measure 200 mL PF254overnight. (35-70 Furthermore, mesh). Fractionation the samples performed powder ofthe NaOH absorbance 0.1 M. valuesA total by of ELISA 100 mLReader solution at λ was405 gradientare inserted using at the a ratio top and of coated chloroform-ethyl with silica transferrednm. Absorbance to microplate values obtained 96 wells are and plotted measure to acetate,gel Merck ethyl 40 PF254 acetate-methanol (35-70 mesh). and Fractionation methanol. ƚŚĞĂďƐŽƌďĂŶĐĞǀĂůƵĞƐďǇ>/^ZĞĂĚĞƌĂƚʄϰϬϱa linear regression equation of standard curve Fractionsperformed obtained gradient accommodated, using a ratio of chloroform-the solvent

The percentage inhibition of haem polymerization calculated by the formula:

΀ȲͲŚĞŵĂƚŝŶ΁negative controlͲ΀ɴͲŚĞŵĂƚŝŶ΁materials testing % Inhibition = x 100%

΀ȲͲŚĞŵĂƚŝŶ΁ negative control

133134 TMJ • Vol. 01 • No.02 • 131-140

evaporated and its profile monitored using thin Therefore, very few in number, the isolates layer chromatography. TLC profile that has a were analyzed by GC-MS. Mass spectrum of fluorescence blue spot found in fractions 10-13 each component compared with mass spectra and then combined into FG8.5 which was isolated available in the Library WILLEY229 GC-MS using preparative layer chromatography (PLC). instrument. Isolation of blue fluorescent compounds in

FG8.5. Isolation of blue fluorescence compounds in FG 8.5 done with PLC by multiple elution. Sample Results and DiscussionS spoted forming elongated ribbon above the plate PLC using the stationary phase silica gel 60 Extraction of Kapur Leaves (H. aculeatus). GF254 Merck with a thickness of 0.5 mm. Plate Extraction of 1.3 kg H. aculeatus leaves powder eluted using a mobile phase of 100% chloroform. was done by maceration with elution graded Making preparative plates by mixing 40 grams of by using n-hexane, ethyl acetate and methanol silica gel 60 GF254 Merck (1.07730.1000) and 80 ml solvents. After the solvent evaporated, 15.6 g of distilled water in a closed erlenmeyer, beaten (1.2%) n-hexane, 53.3 g (4.1%) ethyl acetate and for ± 2 minutes, then spread out on 6 pieces of 61.1 g (4.7%) methanol extracts were obtained. glass plates measuring 20 x 20 cm. Silica plate In Vivo Antiplasmodial Activity Test. Extracts was allowed to dry (± 18-20 hours) and activated were tested on in vivo antiplasmodial activity 0 in an oven at a temperature of 100-110 C for in P. berghei infected mice. The ED50 values ± 60 minutes if want to use. Plates that have were 467.58; 2074.02 and 16.16 mg/kgBW, for been activated, refrigerated until ready to use. n-hexane, ethyl acetate and methanol extracts Sample solution spoted on the elongated ribbon- respectively. Table 1 showed the effect of H. shaped plate and then aerated until all the aculeatus extract on the growth of P. berghei in solvent evaporates. Plate were inserted into the Swiss mice. chamber containing chloroform. After elution is Table 1 showed that the mean parasitemia completed, the plates is taken from chamber and obtained in general decreased with increasing the chloroform evaporated by means aerated dose, except in the group of ethyl acetate extract. to dry. Plate inserted into the chamber again. The greater the dose of ethyl acetate extract, the Elution performed 4 times. Blue fluorescence less parasitemia but at doses of 100 mg/kgBW/d bands scraped and dissolved in chloroform, there was an increase of parasitemia 14.240 ± stired and filtered. The filtrate was dried with 7.125. The ED50 of methanol extract was lower aerated. Isolates were obtained, checked for (16.16 mg/kgBW) than hexane and ethyl acetate purity by TLC using stationary phase silica gel extracts, therefore methanol extracts had

60 GF254 Merck is eluted with chloroform 100%. greater activity than the hexane and ethyl acetate Chromatograms were observed under the UV. extracts to inhibit growth of Plasmodium. Based Identification of Blue Fluorescent on the criteria for antiplasmodial activity in vivo 7 Compounds. Based on TLC profiles, blue according to Munoz et al. , the ED50 ≤ 100 mg/ fluorescence isolate can be estimated that the kgBW/d categorized into very good, ED50 : 101- isolate is not pure. This can be seen that even 250 mg/kgBW/d is good, ED50: 251-500 mg/kgB/d though these isolates seem to give one spot, is moderate and inactive when ED50 > 500 mg/ but revealed distinct blue fluorescence intensity kgBW/d. Thus, the methanol extract (ED50:16.16 when observed under UV 254 nm and 366 nm. mg/kgBW) had very good antiplasmodial

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Table 1. In vivo antiplasmodial activity of H. aculeatus extract in Plasmodium berghei infected Swiss mice

Mean Mean % Extract Dose (mg/kgBW) ED50 (mg/kgBW) Parasitemia ± SD Inhibition ± SD 400 8.45 ± 6.51 46.94 ± 40.92 200 12.51 ± 7.99 21.32 ± 50.30 Hexane Extract 100 13.95 ± 6.36 12.22 ±40.00 467.58 50 15.30 ± 7.57 3.76 ± 47.64 25 19.66 ± 7.61 -23.69 ± 52.86 200 10.75 ± 3.18 17.29 ± 29.89 100 14.24 ± 7.12 32.38 ± 20.03 Ethyl acetate 50 13.15 ± 4.75 10.42 ± 44.82 2072.38 Extract 25 13.15 ± 6.02 17.29 ± 37.89 12,5 14.89 ± 6.58 6.33 ± 41.40 80 5.79 ± 2.80 63.59 ± 17.62 40 6.86 ± 1.06 56.83 ± 6.64 20 7.05 ± 6.80 55.63± 42.78 Methanol Extract 10 8.31 ± 6.93 47.71 ± 43.59 16.16 5 9.59 ± 2.21 39.66 ± 13.93 2,5 10.70 ± 5.64 32.68 ± 35.49 1,25 12.40 ± 6.02 22.01 ± 37.84 Negative control 0 15.90 ± 5.84 0.00 ± 0.00

11 activity,250 mg/kgBW/d the ethyl is good, acetate ED 50 extract: 251-500 was mg/kgB/d inactive extract of sungkaipasak bumi leaf (EDand50 methanol= 87.79 mg/kgBW) extract of 12 is(2074.02 moderate mg/kgBW) and inactive and when hexane ED 50 extract > 500 mg/ was meniranand pauh have kijang an in leaf vivo (ED antiplasmodial50:36.95 mg/kgBW) activity. moderatekgBW/d. Thus, (467.58 the methanolmg/kgBW). extract Methanol (ED50:16.16 extract moreBased potential on the ED than50 value kapur of leaves. this some medicinal hasmg/kgBW) active compounds had very that good could antiplasmodial inhibit the plants,Those the data ethanol supports extract the truth of mimba of the leaves,use of growthactivity, ofthe Plasmodium ethyl acetate compared extract withwas inactive hexane kapurmethanol leaves extract empirically of pasak by the bumi people and ofmethanol Maluku and(2074.02 ethyl mg/kgBW) acetate extract. and hexane Some extract medicinal was asextract an antimalaria. of meniran Althoughhave an in certain vivo antiplasmodial compounds plantsmoderate of Indonesia(467.58 mg/kgBW). that has beenMethanol reported extract to thatactivity could more inhibit potential Plasmodium than kapur from leaves. methanol havehas activeantiplasmodial compounds activity that in could vivo are inhibit ethanol the extractsThose of data kapur supports leaves have the nottruth been of theidentified, use of 8 extractgrowth of of mimbaPlasmodium leaves compared (ED50:1.27 withmg/kgBW) hexane, expectedkapur leaves potential empirically of by the the methanol people of Maluku extract methanoland ethyl extract acetate of extract. the roots Some of pasak medicinal bumi ofas an kapur antimalaria. leaves can Although be used certain as a referencecompounds to

(EDplants50:11.20 of Indonesia mg/kgBW), that brotowali has been (ED 50 reported:97.04 mg/ to searchthat could and inhibit develope Plasmodium a new antimalarial from methanol drugs 9 kgBW)have antiplasmodial, meniran (ED 50activity:9.1 mg/kgBW), in vivo are mahogany ethanol fromextracts natural of kapur medicinal leaves plantshave not been identified, 10 8 (EDextract50:199.87 of mimba mg/kgBW) leaves (ED, aqueous50:1.27 mg/kgBW) extract of, expectedFractionation potential of of Methanol the methanol Extract extract of H. 11 sungkaimethanol leaf extract (ED50 = of 87.79 the mg/kgBW) roots of pasak and bumipauh aculeatusof kapur leaves l e ave s . can Vacuum be used liquid as chromatography a reference to 12 (EDkijang50:11.20 leaf (ED mg/kgBW),50:36.95 mg/kgBW) brotowali (ED50:97.04. Based on columnsearch and was develope used a for new fractionation. antimalarial drugs The 9 themg/kgBW) ED50 value, meniran of this some (ED50 :9.1 medicinal mg/kgBW), plants, separationfrom natural is medicinal based on plants differential partitioning 10 mahoganythe ethanol (EDextract50:199.87 of mimba mg/kgBW) leaves,, methanol aqueous betweenFractionation the mobile of Methanol and stationary Extract phases. of H.

135136 70-9RO1RTMJ • Vol. 01 • No.02 • 131-140 aculeatus l e ave s . Vacuum liquid chromatography Table 2. The IC50 HPIA of combined fraction of Thecolumn stationary was usedphase usedfor fractionation. was silica gel 60H The H. aculeatus methanol extracts (1105932)separation and is based the mobile on differential phase using partitioning a variety Combined ofbetween ratio n -hexane-chloroform the mobile and stationary and chloroform- phases. Mean of IC50 ± SD (µg/ml) methanolThe stationary and methanol phase used in gradient. was silica A gelnumber 60H fraction of(1105932) 62 fraction and wasthe mobileobtained phase from using fractionation a variety FG 1 38217.88 ± 66171.37 of ratiomethanol n-hexane-chloroform extract and based and on chloroform- thin layer FG 2 1E+0.10 ± 1.732E+0.10 chromatography,methanol and methanol 18 in (FG gradient.1-FG18) A combined number FG 3 1229.78 ± 543.12 fractions,of 62 fraction similar was chromatograms obtained from fractionation profiles was FG 4 536.38 ± 286.58 obtained.of methanol Those extract combined and fractions based on were thin tested layer FG 5 1258.25 ± 126.36 onchromatography, heme polymerization 18 (FG inhibitory-FG ) activity combined to 1 18 FG 6 170.26 ± 135.96 fractions,identify the similar most active chromatograms one profiles was FG 7 137.12 ± 103.54 obtained.Haem Those Polymerization combined fractions Inhibition were Activity tested FG 8 18.22 ± 7.25 onTest heme of Combinedpolymerization Fraction inhibitory of activity Methanol to FG 9 859.67 ± 1116.48 identifyExtracts the. Haem most polymerization active one is a mechanism in changing Haem Ferrriprotoporfirin Polymerization IX (FPIX) Inhibition which FG 10 3E+011 ± 5.774E+011 Activityis toxic Test to Plasmodium of Combined. HaemFraction polymerization of Methanol FG 11 2E+038 ± 4.04E+0.38 Extractsoccurs within. Haem the polymerization food vacuole ofis P.a mechanismfalciparum. FG 12 18165.84 ± 29569.35 FPIXin changing formed Ferrriprotoporfirin when Plasmodium IX (FPIX) degrade which FG 13 9601.45 ± 13707.42 hemoglobinis toxic to Plasmodium as a source. Haem of nutrients. polymerization FPIX be FG 14 594313.7 ± 1020187 polymerizedoccurs within into the hemozoin,food vacuole a non of P.toxic falciparum malarial. FG 15 22145.13 ± 31317.80 pigment.FPIX formed Hemozoin when is identicPlasmodium with β-hematin. degrade FG 16 2590962 ± 8961.30 Haemhemoglobin polymerization as a source inhibitory of nutrients. activity FPIX of be a FG 17 9871.13 ± 8961.30 compoundpolymerized is into directly hemozoin, related a tonon its toxic potential malarial as FG 18 1401.69 ± 899.14 anƉŝŐŵĞŶƚ͘ antimalaria. ,ĞŵŽǌŽŝŶ Concentration ŝƐ ŝĚĞŶƚŝĐ of ǁŝƚŚ β-hematin ɴͲŚĞŵĂƚŝŶ͘ from P o s i t i v e c o n t r o l 240.98 ± 50.14 compoundHaem polymerization tested and inhibitory control samples activity of were a (chloroquine) calculatedcompound basedis directly on standard related tocurve its potentialequation: as y =ĂŶĂŶƚŝŵĂůĂƌŝĂ͘ŽŶĐĞŶƚƌĂƚŝŽŶŽĨɴͲŚĞŵĂƚŝŶĨƌŽŵ 0.378 x + 0.166, y was absorbance value and xcompound was concentration tested and of β-hematin. control samples The value were of (fractionchloroquine. 5, 6 and Haem 7) of polymerization kembang bulan inhibitoryleaves (T. 2 Rcalculated was 0.969. based Probit on analysisstandard showed curve equation:that IC50 of y diversifoliaactivity had) methanol been extract reported and from the IC50 several were FG= 0.378 varies x between+ 0.166, y0.018 was -absorbance 3x107 mg/ml value and andthe medicinal162.20, 216.30, plants and such 231.54 as ether µg/ml soluble respectively fraction13. positiveǆǁĂƐĐŽŶĐĞŶƚƌĂƚŝŽŶŽĨɴͲŚĞŵĂƚŝŶ͘dŚĞǀĂůƵĞŽĨ control (chloroquine) was 240.98 µg/ml The(fraction FG8 methanol5, 6 and 7) extract of kembang of kapur bulan leaves leaves had (T. 2 (TableR was 2).0.969. Probit analysis showed that IC50 of ICdiversifolia50 lower ) thanmethanol those extract extracts. and Thisthe IC50 indicates were 7 13 FG variesAmong between the 18 FG,0.018 the - 3x10most mg/mlactive fractionand the 162.20,that the 216.30, FG8 methanol and 231.54 extract µg/ml of respectively kapur leaves. inpositive inhibiting control haem (chloroquine) polymerization was 240.98was FG µg/ml8 due haveThe FGgreater8 methanol activity. extract of kapur leaves had to(Table the 2). smallest IC50 value that was equal to IC50 Phytochemicals lower than those Active extracts. Fraction This (Combined indicates

18.22Among ± 7.25 the µg/ml. 18 FG, When the most compared active withfraction IC50 Fractionthat the 8) FG of8 methanol Methanol extract Extracts of H. kapur aculeatus leaves. valuein inhibiting of positive haem control polymerization (chloroquine), was FG8FG8 duehad Phytochemicalhave greater activity. analysis of FG8 methanol extracts lowerto the value smallest showed IC50 that value activity that of was FG 8 equalon haem to of PhytochemicalsH. aculeatus leaves Active usingFraction several (Combined spray polymerization18.22 ± 7.25 µg/ml. inhibitory When compared was greater with than IC50 Fractionreagents such8) of asMethanol dragendorf, Extracts anisaldehid H. aculeatus sulfate,. chloroquine.value of positive Haem control polymerization (chloroquine), inhibitory FG8 had Phytochemicalsitroborat, Liebermann-Burchard, analysis of FG8 methanol FeCl extracts3 and loweractivity value had showed been that reported activity of from FG8 on several haem KOH.of H. Three aculeatus groups leaves of triterpenoid using several compounds, spray polymerizationmedicinal plants inhibitorysuch as ether was soluble greater fraction than phenolicreagents such compounds as dragendorf, and essential anisaldehid oils sulfate, were

136137 Rachel Turalely et.al, In vivo Antiplasmodial of the Most Active Fraction and Its Compound of Kapur Leaves (Harmsiopanax aculeatus Harms) Extract Against Plasmodium berghei

obrtained. did not seem to separate. After the band of blue

Fractionation Combined Fraction 8 (FG8). fluorescence at 365 nm UV marked, scraped

The most active fractions (FG8) from methanol and then extracted with chloroform, the filtrate extract further separated using pressed column evaporated in a way aerated. Isolates were then chromatography using stationary phase of silica analyzed using the blue fluorescence GC-MS. gel 60 PF254 Merck (230-400 mesh) and a mobile GC-MS analysis of Isolates Blue Fluorescent. phase gradient using chloroform-ethyl acetate GC-MS analysis of the isolates showed that the and ethyl acetate-methanol at various ratio. blue fluorescence compound was not a single Fraction of its similar TLC profiles were merged compound but contain many compounds. and 19 fractions were obtained. Fractions 10- More than 3.11% of the amount consist of 7

13 were grouped in FG8.5 show a dominant blue compounds such as essential oils (eugenol), fluorescence (Figure 1). Further separation was straight-chain saturated fatty acids and esters intended to isolate the fraction of compounds ( isopropyl myristate, methyl ester of palmitate that show blue fluorescence. and palmitic acid) and essential oil (eugenol), straight chain saturated fatty acids and esters (isopropyl myristate, methyl ester of palmitic acid and palmitic), phtalat acid ester such as 1,2 Benzendicarboxylic acid bis (2-methylpropyl) ester, 1,2 Benzendicarboxylic acid Butil 2-metilpropil ester, and 1,2 Benzendicarboxylic acid bis (2-ethylhexyl) ester. According to some research, palmitic acid can induce cardiomyocyte apoptosis through de novo ceramide formation and activation of mitochondria14; to regulate apoptosis, such as in the case of pancreatic cells and induces Figure 1. TLC profile of FG8.5 methanol extract cell death through mtDNA and genomic DNA of H. aculeatus leaves damaged15. It was also reported that palmitic acid is antibacterial, antitumor and antifungal16,17,18. Isolation Blue fluorescence compounds in Fatty acids that have been reported as an the FG8.5. Blue fluorescence compounds in FG8.5 antimalarial is oleic fatty acids and showed a very methanol extracts of H. Aculeatus leaves isolated weak activity in vitro1. 1,2 benzendicarboxylic using preparative thin layer chromatography (PLC) acid bis (2-ethylhexyl) ester reported to have by multiple elution (4 times elution) with 100% activity as an antibacterial, can inhibit tumor chloroform eluent. Elution results showed there growth and potential as an antifungal19. Eugenol were 4 bands at UV 254 nm and 3 bands at 365. is widely used in the medical field (dentistry) PLC chromatogram showed that the third band as well as pharmaceutical. Hemaiswarya and (UV 254 and 365 nm ), had blue fluorescence. Doble20, reported that eugenol has a biological Fluorescence bands at UV 365 nm showed activity as antibacterial against gram-positive thicker than UV 254 nm. The blue fluorescence bacteria. Although many studies have reported compounds contain many compounds with on various biological activities of palmitic acid; similar polarity, so that in the chromatogram (2-ethylhexyl) pthalat and eugenol, but its activity

138 TMJ • Vol. 01 • No.02 • 131-140

as an antimalarial not yet known and needs to Acknowledgement: be examined further. It is estimated that seven of these compounds may have antimalarial This study received financial support from activity. According Bassilico et al.6, compounds Level I Government of Maluku Province and Dr. that can inhibit the polymerization of haem Hilda Ismail, MSi, Apt. The authors wish to thank are the compounds which have OH groups Mr. Anto and Mr. Purwono for the excellent which can react with iron ions in the haem so technical assistance. that it can inhibit the polymerization of haem. Eugenol and palmitic acids have hydroxyl groups as well as fatty acid ester hydrolyse reaction to References: form fatty acids and alcohols which contain OH groups. Phthalat esters also hydrolyse reaction 1. Tringali C. Bioactive Compound from Natural to form a carboxylic acid and alcohol containing Sources. Taylor & Francis, London and New OH groups. Thus the seven compounds were York, 2001. expected as potential antimalarial candidate. 2. Center for Disease Control and Prevention, Malaria. 2010. Available from: http://www. cdc.gov/malaria/html. [cited: May,31- 2010]. Conclusion 3. Harijanto PN. MALARIA. Epidemiologi, Patogenesis, Manifestasi Klinik, & Based on this results, it can be concluded that Penanganan. Jakarta: EGC, 2000: 36, 152-3. methanol extract of kapur leaves (H. aculeatus) 4. Perez H, Diaz F, Medina JD. Chemical was the most potential in vivo antiplasmodial investigation and in vitro antimalarial activity activity with ED50 value of 16.16 mg/kgBW. of Taebuina ochracea ssp. neochrysantha. Combined fraction 8 was the most active IJP, 1997. 35(4): 227-31. fraction with IC50 HPIA of 18.217 ± 7.246 µg/ml. 5. Peters WJ, Portus JH. & Robinson BL. The The content of compounds in methanol extracts chemotherapy of rodent malaria XXII. The

FG8 were triterpenoids, phenolic compounds value of drug-resistant strains of P.berghei and essential oils. Seven blue fluorescence in screening for blood schizontisidal activity. compounds in FG8.5 identified using GC-MS were Ann.Trop.Med.Parasitol, 1975: 69:155-71. essential oils (eugenol), straight-chain saturated 6. Bassilico N, Pagani E, Monti D, Olliaro P and fatty acids and esters (isopropyl myristate, Taramelli D. A Microtitre based Method methyl ester of palmitate and palmitic acid) and for Measuring the Haem Polimerization essential oil (eugenol), straight chain saturated Inhibitory Activity (HPIA) of Antimalarial fatty acids and esters (isopropyl myristate, Drugs. J. Antimicrob. Chemother, 1998: 42: methyl ester of palmitic acid and palmitic), 55-60. phtalat acid ester such as bis(2-methylpropyl) 7. Munoz V, Sauvalin M, Bourdy G, Callapa J, phtalat, Butil 2-metilpropil pthalat, and bis(2- Bergeron S, Rojas I, Bravo JA, Balderrama ethylhexyl) phtalat. L, Ortiz B, Gimenez A, Deharo E. A Search

139 Rachel Turalely et.al, In vivo Antiplasmodial of the Most Active Fraction and Its Compound of Kapur Leaves (Harmsiopanax aculeatus Harms) Extract Against Plasmodium berghei

for Natural Bioactive Compounds in Bolivia 14. Dyantar D, Eberhardt ME, Maedler K, Through a Multidisciplinary Activity of Pruschy M, Eppenberger HM, Spinas GA, Plants used by the Chocobo Indians. J. Donath MY. Glucose and Palmitic Acid Induce Ethnopharmacol, 1999: 1-11. Degeneration of Myofibrils and Modulate 8. Muhtadi. The Separation of Compounds Apoptosis in Rat Adult cardiomyocytes. J. and Fraction Virtuing Antiplasmodium Diabetes, 2001:50: 2105-13. From Methanol Extracts of Mimba Tree Bar 15. Zeng L, Wu GZ, Goh KJ, Lee YM, Ng CC, You (Azadirachta indica Juss). J. Penelitian Sains AB, Wang J, Jia D, Hao A, Yu Q, Li B. Saturated & Teknologi, 2008. Vol. 09 (2): 117-36. Fatty Acids Modulate Cell Response to 9. Qamariah N. Aktivitas antiplasmodial in vitro DNA Damage: Implication for Their Role in dan in vivo ekstrak air Eurycoma longifolia Tumorgenesis. PLos One, 2008: 3(6): e2329. Jack., Tinospora tuberculata, Jack., Swietenia 16. Yu FR, Lian XZ, Guo HY, McGuire PM, Li RD, mahagoni, Jack., Azadirachta indica A. Jus. Wang R, Yu FH. Isolation and Characterization [Tesis]. Yogyakarta, Program Pascasarjana of methyl esters and derivates from Universitas Gadjah Mada, 2002. Euphorbia kansui (Euphorbiaceae) and their 10. Mustofa, Sholikah EN, Wahyuono S. In inhibitory effects on the human SGC-7901 vitro and In vivo Antiplasmodial Activity and cells. J. Pharm. Pharmaceut Sci, 2005: 8(3): Cytotoxity of Extracts of Phyllanthus niruri L. 528-35. Herbs Traditionally Used to Treat Malaria in 17. Bazes A, Silkina A, Douzenel P, Fay F, Kervarec Indonesia. J. Trop Med Public Health, 2007: N, Morin D, Berge JP, Bourgougnon N. 38(4): 609-15. Investigation of antifouling constituents from 11. Suwandi JF. Aktivitas Antiplasmodium Ekstrak the brown alga Sargassum muticum (Yendo) Daun Sungkai (Peronema canescens): Kajian Fensholt. J. Appl. Phycology, 2009: 21(4): aktivitas antiplasmodium in vitro dan in vivo, 395-403. aktivitas penghambatan polimerisasi hem 18. Ogunlesi M, Okiei W, Osibote AA. Analysis of dan aktivitas sitotoksik terhadap sel vero. the essential oil from the leaves of Sesamum [Tesis]. Yogyakarta, Universitas Gadjah Mada, radiatum, a potential medication for male 2007 infertility factor, by gas chromatography- 12. Praptiwi Chairul. Pengaruh Pemberian mass spectrometry. African J. of Biotech, Ekstrak Pauh Kijang (Irvingia malayana Olive 2010: 9(7): 1060-67. ex A. Benn) Terhadap Tingkat Penurunan 19. Shanab SMM, Shalaby EA, Lightfoot DA, El- Parasitemia pada Mencit yang Diinfeksi Shemy HA. Allelopathic Effects of water Plasmodium berghei. J. Biodiversitas, 2008: Hyacinth [Eichhornia crassipes]. J.PLoS One, 9(2): 96-8. 2010: 5(10): e13200. 13. Syarif RA. Aktivitas Antiplasmodium Fraksi 20. Hemaiswarya S, Doble M. Synergistic Larut Eter Ekstrak Metanol Daun Kembang interaction of eugenol with antibiotics Bulan (Tithonia diversifolia (Hemsley) A. against gram negative bacteria. J. Phytomed, Gray) Pada Plasmodium falciparum secara in 2009: 16(11): 997-1005. vitro. [Tesis]. Yogyakarta, Universitas Gadjah Mada, 2007.

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