BIOASSAY-GUIDED FRACTINATION OF ANTIMITOTIC COMPOUND FROM ONGKEA CORTEX ( MEZZETTIA PARVIFLORA BECC ) TOWARDS SEA URCHIN EGGS

Ongkea cortex, the wood bark of Mezzettia Parviflora Becc, is a traditional medicine originated from Southeastern Sulawesi (Indonesia). It has been empirically known to have antitumor property. In this study, we examined the antiproliferative activity and obtained the antimitotic compound of ongkea cortex. Antimito tic activity was ultimately determined by the inhibition of cleavage-stage of newly fertil ized sea urchin ( Lytechinus variegatus ) eggs. A bioassay-guided fractination was performed in order to find the bioactive substance of ongkea cortex. The IC 50 values of methanolic extract, ethyl acetate-solubl e part of metanolic extract and ethil acetat insolubl e part of metanolic extract were 1221.68 μg/mL, 2.69 μg/mL, and 15.15 μg/mL, respectively. Ethyl acetate-soluble part of metanolic extract was further investigated. It was partitionated using vacuum liquid column chromatoghraphy with different solvent syste m by increasing their polarities. There were three different fractions obtained. Frac tion III exerted the highest inhibition activity with IC50 value of 1.33 μg/mL. It was separated subsequently to result four groups of compounds. III-C group presented the most potent inhibition activity with IC50 value of 0.7147 μg/mL. It was then subjected to preparative TLC and yieldedsix groups of subfractions. III-C-3 subfraction was indicated as the most potent compound with IC 50 value of 0.3378 μg/mL. It was ten times weaker compared with antimit otic activity of Vincristine with IC50 of 0.0351 μg/mL. As a conclusian, ongkea cortex might have antimitotic property with the highest rate inhibiti on activity exhibited by III-C-3


INTRODUCTION
Ongkea cortex (Mezzettia Parviflora Becc.), belongs to annonaceae family, is a traditional substance that has been empirically used in Southeast Sulawesi (Indonesia) to treat some diseases such as hipercholesterolemia, asthma, diabetes mellitus and tumor [1].
The familiy of annonacea has a broad range of pharmacological activities such as antimicrobial, antifungal, antitumor, and insecticidal properties.It has some various chemical compounds such as benzylisoquinoline, acetogenin, c-benzil-flavonoid and diterpenoid alkaloids [2].Based on the concept of chemotaxonomy, chemical compounds of plants from the same familial groups are mostly similar.
Previous studies have reported that ongkea cortex has antiatherosclerotic, antiplatelet and free radical scavenging activities [3,4,5].Yet, there is no research that has been performed to investigate its antimitotic property.Therefore, the present work is the first study trying to evaluate the possibility of ongkea cortex to be developed as an anticancer agent.
To analyse theantiproliferative activity of ongkea cortex, we used an inhibition of sea urchin (Lytechinus variegates) eggs development assay.It is a proper bioassay model to study biological activities such as antimitotic, teratogenic and antineoplastic activities [6].
In this study, the antitumor activity of ongkea cortex was evaluated to provide scientific based information as a traditional medicine.A bioassay-guided fractination was performed in order to find the bioactive compound of ongkea cortex.

METODE PENELITIAN Samples Collection
Ongkea cortex was obtained from Buton regency, Southeast Sulawesi.Samples were cleaned, shattered, dried and powdered.

Samples Extraction
Dried powdered portions of 1.6 Kg of ongkea cortex were extracted by maceration method with 12 Litres of methanol for 3x3x24 h.The results were collected and rotoevaporated to yield viscous methanol extract.

Partition of methanol extract
Methanol extract was partitionated with ethil acetate using liquid-solid partition method.It produced ethyl acetate-soluble part of metanolic extract and ethil acetat insoluble part of metanolic extract.The chemical compound profiles of extracts were monitored by thin layer chromatography.Antimitotic activities of extracts were investigated by an inhibition of sea urchin (Lytechinus variegates) eggs development assay.

Determination of Antiproliferative Activity
Antimitotic effect was determined by the ability of samples to prevent the cleavage-stage of newly fertilized sea urchin (Lytechinus variegatus) eggs.The procedure was based on the study demostrated by Jiminez [7].Gamete elimination was induced by injecting 1 mL KCl 10 % in the perivisceral cavity.Fertilization was undertaken by mixing 1 mL of a sperm suspension and 4 mL of eggs in the chamber filled with 50 mL of filtered seawater.Methanolic extract, ethyl acetate-soluble part of metanolic extract and ethil acetat insoluble part of metanolic extract plus filtered sea water were added to 100 µl of fertilized eggs in the eppendorf to make concentrations 1, 10 and 100 ppm in a final volume of 1 mL.The mixtures were incubated at 15-20 °C with intermitten shaking.The observation of dividing cells was carried out after 2 h incubation.Filtered seawater and DMSO were used as negative controls.Vincristine (0.01 ppm, 0.1 ppm, and 1 ppm) was used as a positive controls.Experiment was carried out in triplicate.The same examination was applied to test the activities of fractions but with different series concentration i.e. 0.2, 1.5, 25 and 125 ppm.The number of divided and non divided cells were counted, analyzed, and displayed with IC 50 (Inhibitory Concentration 50%).

RESULTS AND DISCUSSSION
The antiproliferative assay using sea urchin eggs has been well known to have a good sensitivity against toxic substance.It can help to assess the ability of natural compounds to be developed as an anticancer.The inhibition of growth cycle of sea urchin eggs can be associated with several steps such as DNA and RNA synthesis, protein synthesis, and mitotic spindle assembly [8].
A first screening showed that the IC 50 values of methanolic extract, ethyl acetate-soluble part of metanolic extract and ethil acetat insoluble part of metanolic extract were 1221.68 µg/mL, 2.69 µg/mL, and 15.15 µg/mL, respectively (Table 1).Ethyl acetatesoluble part of metanolic extract possesed the highest inhibition rate.Figure 1 showed its effect on the initial development of sea urchin embryos.In order to find the active principles possibly responsible for antimitotic activity of ongkea cortex, ethyl acetatesoluble part of metanolic extract was further investigated by bioassay-guided fractination.It was partitionated using vacuum liquid column chromatoghraphy with different solvent system by increasing their polarities yielded three different group of bioactive fractions.Fraction III exerted the highest inhibition activity with IC 50 value of 1.33 µg/mL while Fraction I and II had IC 50 of 7.39 µg/ml and 7.48 µg/ml, respectively (Table 2).The inhibition effect of fraction III on sea urchin eggs was demonstrated by figure 2.
Fraction III was separated subsequently to result four groups of compounds that were then exposed to sea urchin eggs.Fraction III-A, III-B, III-C and III-D exerted high toxicity activities with IC 50 1.2971µg/mL; 1.9480 µg/mL; 0.7147 µg/mL; and 3.2569 µg/mL, consecutively (Table 3).The most bioactive substance was seemingly in Fraction III-C.Furthermore, it was subjected to preparative TLC and yielded six groups of subfractions.Their IC 50 were 3.4411 µg/mL; 3.7153 µg/mL; 0.3378 µg/mL; 59.827 µg/mL; 85.2903 µg/mL; 40,794.3483µg/mL for III-C-1, III-C-2, III-C-3, III-C-4, III-C-5, and III-C-6, respectively (Table 4).Among them, subfraction III-C-3 exhibited the most potent compounds.Although, it was ten times weaker compared with antimitotic activity of Vincristine with IC 50 of 0.0351 µg/mL.Negative controls, filtered water and DMSO, had no inhibition effect.Jacobs reported that if there are compounds presenting 100% inhibition in sea urchin eggs assay at a concentration 16 µg/mL or less, they should be very potential to be investigated as anticancer agents in in vivo experiment [9].Based on this information, it can be stated that subfraction III-C-3 has a strong antiproliferative activity.Therefore, the potency of subfraction III-C-3 of ongkea cortex as a natural antimitotic agent and its mechanisms should be further elaborated.

CONCLUSION
Finally, it can be concluded that ongkea cortex might have potent antitumoral property with the highest rate inhibition activity presented by III-C-3 compound.We suggest to evaluate the cytotoxicity effect of III-C-3 compound using human cell lines and in vivo experiment with animal model.

Table 1 .
Antiproliferative activity on sea urchin egg development of methanolic extract, ethyl acetate-soluble part of metanolic extract and ethil acetat insoluble part of metanolic extract of ongkea cortex

Table 2 .
Antiproliferative activity on sea urchin egg development of Fraction 1,II and III.