Kerjasama penelitian Unhas-UGMThe emergence of drug resisntant and multi drug resistant TB and malaria strains, as well as the lack of any current chemoteraphy augmented the necessity to serach for new and better anti_TB, malaria, toxoplasmosis and other anti-infective drug leads. Polar fraction of acetone extract of BL-10 was considered to be potential compounds for further characterization as anti infective agents

Astuti, Puji

Hertiani, Triana

Tahir, Akbar

Utami, Sylvia Tanjung

Wahyuono, Subagus

Hasanuddin University Repository

Marine Sponge Jaspis sp, A Potential Bioactive Natural Source 
against Infectious diseases 
Fuji Astuti 1, Sylvia Tunjung Utami 1, Triana Hertiani 1, Gemini Alam 2, Akbar Tahir 3, 
Subagus Wahyuono 1 
1
 Faculty of Pharmacy, Gadjah Mada University, Yogyakarta 
2
 Department of Pharmacy, h acuity of Natural Science, University of Hassanudin, Makasar 
Coral Research Center, University of Hassanudin, Makasar 
ABSTRACT 
Puji Astuti, Sylvia Tunjung Utami, Triana Hertiani, Gemini Alam, Akbar Tahir, Subagus Wahyuono - Marine 
Sponge Jaspis sp, A Potential Bioactive Natural Source against Infectious diseases 
Background: The emergence of drug resistant and multidrug-resistant TB and malaria strains as well as the lack of 
any current chemotherapy augmented the necessity to search for new and better anti-TB, malaria, toxoplasmosis and 
other anti-infective drug leads 
Objective: This study aims to screen potential anti infective extracts collected from Barrang Lompo island and 
report on their antibacterial and antifungal properties. 
Methods: Testing for anti-infective agents was conducted using dilution method. Nutrient Agar was used as the 
testing media and nutrient broth for the inoculation of microorganisms. Staphylococcus aureus, Escherichia coli and 
Salmonella thipii were used as the testing bacteria and Candida albicans for the testing fungi. Chloramfenicol was 
used as positive control for antibacterial testing and ketocozaole for anti fungal testing. 
Results: From the 11 acetone extracts tested. BL-02. BL-09. BL-iO and BL-12 was found to inhibit the erowth of 
microorganisms and the extract of BL-10 was found to be the most active. Autobiography results suggest that the 
polar fractions were responsible for the growth inhibition 
Conclusion the polar fraction of acetone extract of BL-10 was considered to be potential compounds for further 
characterization as anti infective agents. 
Key words: screening, sponge, anti bacteria, anti fungi 
ABSTRAK 
Puji Astuti, Sylvia Tunjung Utami, Triana Hertiani, Gemini Alam, Akbar Tahir, Subagus Wahyuono - Spons Jaspis 
sp, Sumber Alam Bioaktif yang Potensial Melawan Penyakit Infeksi 
Latar Belakang: Meningkatnya kasus resistensi obat dan resistensi kembinasi obat untuk TB dan malaria serta 
terbatasnya obat kemoterapi yang baru menunjukkan pentingnya penelitian terhadap senyawa yang baru dan lebih 
baik serta berpotensi sebagai anti-TB, malaria, toxoplasmosis dan penyakit infeksi lainnya. 
Tujuan: Penelitian ini bertujuan untuk skrining ekstraks yang berpotensial sebagai anti infeksi yang dikoleksi dari 
pulau Barrang Lompo serta melaporkan aktivitas anti bakteri dan anti jamurnya 
Bahan dan cara: Uji anti infeksi dilakukan dengan m.etode dilusi padat. Nutrient Agar digunakan sebagai media uji 
sedangkan nutrient broth sebagai media inokulasi mikroorgankme Staphylococcus aureus, Escherichia coli Han 
Salmonella thipii digunakan sebagai bakteria uji dan Candida albicans sebagai jamur uji. Kloramfeniko! digunakan 
sebagai kontro! positif antibakteri dan ketokonazo! untuk antijamur. 
Hasil: Dari sebelas ekstrak yang diuji, empat ekstrak (BL-02, BL-09, BL-10 and BL-12) menghambat pertumbuhan 
bahwa fraksi vang herfanppiina iawah terhadan nenphambatan adalah fraksi vantJ memnnnvai nolaritas tinfwi 
Simpulan: Fraksi polar dari ekstrak aseton BL-10 berpotensial sebagai kandidat eksplorasi lebih lanjut untuk 
penyakit infeksi. 
Kata Kunci: skrining, spons, anti bakteri, anti jamur 
! 
INTRODUCTION 
The rise in the number of immunocompromised patients has led to an increase in bacteria and 
fungal infections. As microbial resistance to classical antibiotics has emerged continuously, an 
urgent demand for a new class of anti infectives has been formulated. Beside the antibiotic 
resistance of bacteria, the non-susceptibiiity of fungal pathogens to classical drugs has become an 
increasing problems, particularly because currently available antimycotics are directed against the 
same limited number of targets within the fungi * . 
u i u n u u u c u u i i u l U i u v C l a . i i i i i i i O l i u v u i l u v u v i i js^cu v v U i i u v v i u v vvi l i i i i i i i u i J i i v a o v j 
reported in 1998. The most dangerous malaria parasite, Plasmodium faliparum which causes 
cerebral malaria, is expected to spread in the central or northern regions of Europe and North 
America within a few decades . The pathogenic synergy with HIV has increased the overall 
incidence of many opportunistic infectious diseases. For example, the incidence of tuberculosis 
infection in HIV positive cases is 50-fold over HIV negative ones and the same holds true in the 
case of T.gondii4*. The emergence of drug resistant and multidrug-resistant TB and malaria 
strains as well as the lack of any current chemotherapy augmented the necessity to search for new 
and better anti-TB, malaria and toxoplasmosis drug leads 3 , 4 5 
Analysis of the Philippine marine sponge Xestospongia ashmorica afforded four new 
manzamines compounds and reported to have antibacterial properties \ Manzamin A which 
previously received considerable interest because of its potential as anti cancer agents, inhibiting 
malaria parasites not only in vitro but also in vivo1. 
In this paper we screen potential anti infective extracts collected from Barrang Lompo 
island and report on their antibacterial and antifungal properties. 
MATERIALS AND METHODS 
Materials 
The main source of sponges were collected from Barrang Lompo island, the extract obtained 
were designated as BL-01 - BL-12; Nutrient Agar (Difco) was used as the testing media and 
nutrient broth for the inoculation of microorganisms. Staphylococcus aureus, Escherichia coli 
and Salmonella thipii were used as the testing bacteria and Candida albicans for the testing 
2 
fungi. Chlorarnfenico! was used as positive control for antibacterial testing and ketoconazole for 
Methods 
The extracts are obtained by maceration using acetone pa (E.Merck). The solvent was evaporated 
igal testing 
Testing for anti-mfective agents was conducted using dilution method according to Mitscher et al 
(1972). Bacteria and fungi from slant agar were grown in nutrient broth (NB) and incubated for 
j _ Z . T i iuuis . n i t vuiui iy w a s u n u i c u m aailut; i^>av i^ \),y /») uuui [He I U I U I U I I ^ aiuiiicu IVJ a ianua iu 
a 
Mc. Farian (10 CFU) was obtained. The extracts were dissolved using DMSO (200 ul) and 
mixed with liquid form of nutrient agar (NA) (final volume of 10 ml). The mixture was poured 
into dishes and let to become solid. 5 ul of diluted colony was put on the solid medium in which 
the area have been marked and divided into four regions. The colony was spread using drigalsky 
(surface plate method). The dishes containing colony were incubated at 37° C for + 24 hours. 
Cliloramfenicol and ketoconazole were used as positive (H ) controls with DMSO as the negative 
(-) one. 
B ioautography 
The most potent extract was examined using bioautography to determine the active fraction based 
on polarity. This was performed by Thin Layer Chromatography (TLC) followed by anti-
bacterial and anti-fungal testing. The active fraction will show zone inhibition on the bacterial or 
fungal growth. 
Species Identification 
Identification of the species of the active marine sponge was conducted in the Faculty of Biology', 
Gadjah Mada University 
RESULTS AND DISCUSSION 
Screening for anti bacterial and anti fungal was performed using dilution method in which the 
t A i i a v i ^ v v w i v l i i i / v w u v v i t u i iuti iwm agai I I K A I I C I uy LIIW a iu V7i o m a i i v u i u i n v u i l y i v i u w . I U W W A i i a w i i i 
were obtained by maceration using acetone with the consideration that this solvent was able to 
extract non polar as well as polar compounds with minimally contaminated by sea salt. 
According to Mitscher et a/ 8, an extract was considered to be active it" it inhibits the growth of 
microorganism by the concentration of < 1000 u,g/mi. In this experiment, to avoid cross-
contamination with the solvent (DMSO), negative control was made by growing the bacteria in 
the presence of DMSO (200 pi) without the extract. Chloramphenicol and ketoconazole were 
used as the positive controls and was given under concentration the same as the extracts, 1000 
ug/ml. 
Tabei 1. Anti bacterial and anti fungal activities of acetone extracts of sponges collected 
Samples type of bacteria or fungi 
S. aureus E.coli •S'. typhi C. albicans 
BL-01 — — — + 
BL-02 ++ — + — 
BL-03 — — — + 
BL-04 + + — + 
BL-05 — — — 
BL-06 + + + + 
BL-07 + — — — 
BL09 — ++ — 
BL-10 ++ ++ ++ ++ 
BL-11 + — — ~ 
BL-12 ++ + — + 
Control (+) 
Chloramphenicol 
Ketoconayole 
++ ++ ++ ++ 
++ 
Control (-) 
DMSO 
— — — — 
Note: ++ = complete inhibition (no growth) 
+ = low activity (partial growth, relative to the control) 
~ = no activity (complete growth, relative to the control) 
4 
By the given concentration (1000 pg/ml), from the 11 extracts tested, BL-10 was found to 
inhibit the growth of positive-gram bacteria (S. aureus), negative-gram bacteria (E. coli, S. thipii) 
as well as Candida albicans (table 1). BL-09 inhibited the growth of S. aureus and S. thipii but 
not E. coli and Candida albicans. Although BL-02 and BL-12 extracts showed growth inhibition 
of positive-gram bacteria, these extracts did not inhibit the growth of negative-gram bacteria. 
Structure of the cell wall of negative-gram bacteria was suggested to influence the permeation of 
iarge molecule into the cell. Besides consisting of peptidoglycan and teicoat acid, the structure of 
negative-gram bacteria contains other three polymers, i.e. lipoprotein, exomembrane and 
liposacharide, whilst positive-gram bacteria consists of peptidoglycan and teicoat acid only9. 
Since the acetone extract of BL-10 was considered as the most active one, this extract was further 
examined for characterization. 
To determine the active fractions/compounds, bioautography was performed. This method is 
able to detect active fractions/compounds based on the polarity. 1 he fractions/compounds of the 
most active extracts were separated by Thin Layer Chromatography (TLC) with appropriate 
mobile and stationary phase. N-hexane: ethyl acetate = 3:1 was found to be the best mobile phase 
with silica gel GF254 as the stationary phase (figure 1). The separated fractions or compounds 
were tested for their anti bacterial or fungal properties. 
Figure 1. TLC profiles (chromatogram) of the acetone extract of BL-10 using detector cerium 
(IV) sulfate 
5 
Figure 2. Inhibition zone of S. aureus growth from the acetone extract of BL-10 as identified by 
bioautography. Zone of inhibition was identified on the Rf of 0.28 and on the base spot of 
chromatogram. 
The polar fraction of BL-10 acetone extract was found to be responsible for the inhibition 
of the growth of microorganisms. Inhibition zone was observed at the Rf of 0.28 and at the base 
spot of chromatogram when the activity was tested using S. aureus (figure 2). Although in the 
preliminary screening this extract inhibited all microorganisms tested, in this experiment no 
inhibition zone was observed when other microorganism were used. Besides the complexity of 
the cell wall of negative-gram bacteria which was considered to play the role, these results 
suggest influence of the concentration of the compounds in the extract. As an extract contains a 
number of compounds which have different quantities, the highest quantity of compound was 
suggested to dominate the activities of others. Another possible reason is that the concentration of 
the sample tested for TLC was too small that unable to inhibit the growth of microorganism 
tested. 
Based on its morphology, the tissue and the specula, the active extract (BL-10) was 
identified as Jaspis sp. 
CONCLUSION 
1. Acetone extract of BL-10 was found to be the most active extract against microorganism 
tested in which the species was further identified as Jaspis sp. 
2. The polar fractions were suggested to be the ones which are responsible for the inhibition 
of the growth of S. aureus. 
3. These fractions were considered to be potential for further characterization as 
antiinfectious drug leads. 
SUGGESTION 
1. Fractionation and isolation are needed to determine the active antiinfectious compounds 
ACKNOWLEDGEMENT 
We thank to Kirlan, S.Si and Jusain Setiadi, S.Si (University of Hassanudin, Makasar) for 
assistance with sample collection. This work was partly supported by DIKS Faculty of Pharmacy, 
Gadjah Mada University, SK. No. 3760/P/FA/2002, Date 1 July 2002. 
REFERENCES 
1. Wout J van't, 1996, Fungal infection and antifungal drugs: has the age of antifungal 
resistance dawned?, Curr. Opin. Infeci. Dis., 9, 63-66. 
2. VVhite T.C., Marr, K.A., Bowden, R.A., 1998, Clinical, cellular and molecular factors that 
contribute to antifungal drug resistance, Clin. Microbiol. Rev., 1, 382-402. 
3. Rogers, D.J, Randolph, S.E., Science, 2000, 289, 1763-1766. 
4. Jones, P.B., Parrish, N.M., Houston, T.A., Stapon, A., Niharika, P.B., Dick, J.D., 
Townsend, C.A.,J. Med. Chem., 2000, 43, 3304-3314. 
5. Khan, A.A., Slifer, T., Araujo, F.G., Remington, J.S., Antimicrob. Agents Chemother., 
1996,40, 1855-1859. 
6. Edrada, R.U., Proksch, P., Wray, V., Witte, L., Muller, W.E.G., Van Soest, R.W.M., 
1996, Four New Bioactive Manzamine-Type Alkaloids from the Philippine Marine 
Sponge Xestospongia ashmorica, J.Nat.Prod., 59, 1056-1060. 
7. Aug, K.K.H., Holmes, M.J., Higa, T., Hamann, M.T.. Kara. U.A.K., 2000, In Vivo 
Antimalarial Activity of the Beta-Carboline Alkaloid Manzamme A, Antimicrobial 
Agents and Chemotheraphy, 1645-1649. 
8. Mitscher, L.A., Leu, R.P., Bathaia, M.S., Wu, W., Beal, J.L., 1972, Lloydia, 35,157. 
9. Jawetz, E., Melnick, J.L., Adelberg, E., 1995, Medical Microbiology, Apleton & Lange, 
New York, 18-22. 
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Marine Sponges Jaspis sp, A Potential Bioactive Natural Sorce against Infectious Diseases

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Marine Sponges Jaspis sp, A Potential Bioactive Natural Sorce against Infectious Diseases

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