[ABSTRAK
Racun duri Acanthaster planci memiliki beragam aktifitas biologi yaitu aktifitaslethal, aktifitas hemolitik, aktifitas myonecrotic, aktifitas pendarahan, peningkatan aktifitaspermeabilitas kapiler, aktifitas edema, aktifitas phospholipase-A2 (PLA2), aktifitas pelepasanhistamin dari mast cell dan aktifitas kardio vaskular. Racun duri Acanthaster plancimengandung phospholipase A2 (PLA2), plancitoxin yang homolog dengan deoxyribonukleaseII pada mamalia dan plancinin peptida antikoagulan.Berbagai penelitian terdahulu membuktikan bahwa racun yang berasal dari berbagaihewan mengandung senyawa yang potensial dikembangkan sebagai bahan antibiotik danterapeutik untuk mengobati suatu penyakit. Dengan potensi aktivitas biologi tersebut racunAcanthaster planci dapat berkontribusi di bidang medis yang bisa menjadi masukan bagipendapatan negara. Efek antimikrobial hasil aktifitas hidrolisis komponen fosfolipidmembran sel mikroba oleh enzim PLA2 dapat bermanfaat bagi pengembangan bahanantibiotik. PLA2 yang dimurnikan dari racun ular memiliki aktifitas antibakteri terhadapStaphylococcus aureus, Proteus vulgaris, Proteus mirabilis, and Burkholderia pseudomallei.Selain itu, PLA2 memiliki aktifitas antiHIV melalui mekanisme penghambatan pelepasanintraseluler protein capsid virus dan diasumsikan PLA2 memblok virus masuk ke dalam selinang sebelum virus tersebut membuka selaputnya dan secara independent memanfaatkankoreseptornya. PLA2 melindungi sel limfosit T manusia dengan memblok virus yangmemiliki selubung luar mengandung fosfolipid.Acanthaster planci merupakan predator yang mengancam populasi karang terutamaketika terjadi peledakan populasi. Pemanfaatan Acanthaster planci untuk produksi PLA2dapat menjadi alternatif produktif upaya pengendalian populasinya sekaligus membuatnyamenjadi lebih berguna. Purifikasi PLA2 racun duri Acanthaster planci telah dilakukan olehShiomi dan koleganya menggunakan rangkaian kolom kromatografi bertingkat, memerlukanbiaya yang relative mahal dan membutuhkan waktu beberapa hari, sehingga dalam penelitianini dikembangkan metode purifikasi yang sederhana dan cepat dengan biaya yang relatifmurah. Hasil penelitian ini diharapkan dapat menjadi masukan bagi upaya pemanfaatanAcanthaster planci untuk menghasilkan PLA2 yang berpeluang dikembangkan sebagai bahanantibakteri dan antiHIV.Penerapan metode percobaan yang dilakukan dalam penelitian ini memberikan hasilsebagai berikut : Proses ekstraksi racun dari jaringan duri Acanthaster planci berlangsung efektifmelalui proses sonikasi pada 20 kHz selama 2x8 menit (intensitas 80% dan output10). Racun yang terekstraksi tertampung dalam larutan 0,01 M bufer fosfat pH 7,0mengandung 0,001 M CaCl2 yang digunakan sebagai media ekstraksi disebut crudevenom. Pengujian secara kualitatif menggunakan darah manusia yang diberi perlakuancrude venom (1:1) memperlihatkan antikoagulasi darah oleh plancinin yangterkandung dalam racun membuktikan keberhasilan proses ekstraksi. Pada awalnyadilakukan pula metode ekstraksi dengan cara duri diblender terlebih dahulu dandilanjutkan dengan disonikasi. Untuk meminimalisir protein kontaminan yang berasal dari jaringan duri dan mempertimbangkan efisiensi maka metode ini kemudian tidakditerapkan.Purifikasi phospholipase A2 racun duri Acanthaster placi dari Ambon-Maluku melaluipengendapan amonium sulfat bertahap pada tingkat kejenuhan 20% terhadap crudevenom yang telah dipanaskan efektif memurnikan PLA2. Hasil elektroforesis SDSPAGEmemperlihatkan isolat PLA2 memiliki satu pita protein sedangkan crudevenom memiliki empat pita protein. Isolat PLA2 yang dihasilkan memiliki aktifitasspesifik 20 kali aktifitas spesifik crude venom. Pemanasan crude venom pada 60oCselama 30 menit yang diikuti dengan sentrifugasi selama 30 menit pada 15.000xg dan4oC memisahkan protein tidak tahan panas dari PLA2. Metode purifikasi ini jugaditerapkan pada racun duri Acanthaster planci dari Sorong-Papua namun belumberhasil. Sedangkan purifikasi PLA2 melalui pengendapan menggunakan etanoldengan tingkat kejenuhan 80% tidak efektif memurnikan PLA2 namun dapatmeningkatkan aktifitasnya menjadi lima kali aktifitas crude venom. Hasil eksperimenini dipublikasikan di International Journal of Pharma and Bio Science Vol 2/issue2/Apr-Jun 2011 and International Journal of Pharma and Bio Science 2012 Oct;3(4):(B) 603-608 Pengujian aktifitas antibakteri menggunakan metode difusi cakram memperlihatkanterbentuknya zona bening disekitar cakram PLA2 pada kultur Staphylococcus aureusyang mengindikasikan bahwa PLA2 racun duri Acanthaster planci memiliki aktifitasantibakteri terhadap Staphylococcus aureus pada dosis 2, 98 mg/ml. Hasil eksperimenini dipublikasikan pada International journal of Pharma and Bio Sciene 2013 Apr;4(2) : (B)1-5 Pengujian aktifitas antiHIV secara kualitatif menggunakan PBMC pasien HIV(ODHA) memperlihatkan terjadinya penurunan intensitas pita protein DNA pada hasilelektroforesis RT-PCR RNA sampel kultur HIV yang diberi perlakuan PLA2.Selanjutnya analisis kuantitatif hasil Green Fluoresence Particle memperlihatkanterjadinya penurunan jumlah sel yang terinfeksi HIV secara signifikan oleh perlakuanPLA2 dari 9,72% menjadi 0,29% yang mengindikasikan PLA2 racun duri Acanthasterplanci memiliki aktifitas antiHIV. Hasil eksperimen ini dipublikasikan pada AsianPacific Journal of Tropical Medicine (2014) 412-420 Biaya purifikasi PLA2 merupakan pembiayaan yang dibayarkan untuk 1) bahan kimiadan peralatan habis pakai, 2) listrik untuk operasional alat, 3) sewa peralatan dan 4)tenaga kerja. Hasil perhitungan biaya isolasi-purifikasi PLA2 menghasilkan nilai Rp.446.192,- per 50 gram duri dengan hasil yang diperoleh adalah 4,622 mg PLA2. Biayapurifikasi PLA2 miniscale yang dilakukan dalam penelitian ini efisien untukditerapkan dimana harga komersial PLA2 racun ular Crotalus amandetus(Worthington, USA) adalah Rp. 590.000 per mg (59.00 US Dolar).Hasil pengolahan data citra satelit tahun (2006) yang diunduh dari website NASApada Juni 2013 memperlihatkan luas areal terumbu karang yang merupakan habitanAcanthaster planci adalah 94,83 hektar. Diperkirakan pada luas areal tersebut terdapat550 individu dewasa dan jumlah yang dapat dimanfaatkan untuk menghasilkan PLA2adalah 20% dari ketersediaannya per bulan.Berdasarkan hasil percobaan tersebut dapat disimpulkan bahwa : Metode sederhana dan cepat dengan biaya operasionil relatif murah melaluipengendapan 20% amonium sulfat terhadap crude venom yang dipanaskan terlebihdahulu efektif memurnikan PLA2 dari racun duri Acanthaster planci dengan tingkatkemurnian dan aktifitas spesifik yang tinggi. Sedangkan metode pengendapanmenggunakan etanol 80% tidak efektif memurnikan PLA2 dari racun duri Acanthasterplanci namun dapat meningkatkan aktivitasnya menjadi 5 kali crude venom. PLA2 racun duri Acanthaster planci memiliki aktifitas antibakteri terhadapStaphylococcus aureus dan aktifitas antiHIV. Biaya miniscale operasional purifikasi PLA2 efisien untuk diterapkan danketersediaan Acanthaster planci di perairan Liang dan pulau Pombo yang dapatdimanfaatkan untuk menghasilkan PLA2 adalah sebesar 20% per bulan.;

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ABSTRACT
Spines venom of Acanthaster planci have various biological activities: lethal activity,hemolytic, myonecrotic, bleeding, increased capillary permeability, edema, phospholipase A2(PLA2), the activity of histamine release from mast cells and cardio vascular activity. Spinesvenom of Acanthaster planci containing phospholipase A2 (PLA2), plancitoxin which ishomologous with mammals deoxyribonuklease II and plancinin anticoagulant peptide.Previous studies prove that the venoms derived from animals contain variouscompounds that are potential to be developed as antibiotic and therapeutic agents to treat adisease. Acanthaster planci spines venom with various potential biological activity maycontribute in the medical field that can be input for the state revenue. Antimicrobial effectresults by hydrolysis activity of PLA2 on microbial cell membrane phospholipids can bebeneficial to the development of antibiotic agent. PLA2 purified from snake venom haveantibacterial activity against Staphylococcus aureus, Proteus vulgaris, Proteus mirabilis, andBurkholderia pseudomallei. In addition, PLA2 has antiHIV activity through inhibition of therelease mechanism of intracellular viral capsid proteins and assumed PLA2 blocking viralentry into host cells before the virus opens membranes and independently utilizekoreseptornya. PLA2 protect human T lymphocytes by blocking viruses that have outersheath containing phospholipids.Acanthaster planci is a predator threatens coral populations, especially when there is aoutbreak population. Acanthaster planci utilization for the production of PLA2 can be aneffort population control productively and make it more useful. Purification of Acanthasterplanci spines venom PLA2 has been done by Shiomi and colleagues by using a series ofchromatography columns which is relatively expensive and takes a few days, so a simple andfast method with a relatively low cost was developed in this study. The results of this studyare expected to be input for utilaization of Acanthaster planci to produce PLA2 that can bedeveloped as antibacterial and antiHIV agents.Experiments method were conducted in this study gave the following results: Venom extraction from the spines of Acanthaster planci was effective through theprocess of sonication at 20 kHz for 2x8 minutes (intensity 80% and 10 outputs).Venom was accumulated in extraction medium solution of 0.01 M phosphate bufferpH 7.0 containing 0.001 M CaCl2 called crude venom. Qualitative tested by usinghuman blood treated with crude venom (1: 1) showed the blood anticoagulation byplancinin contained in the venom, proves the extraction process successfully. At theprevious conducted on a method of extraction, the spines were blended first andfollowed by sonicated. To minimize contaminant proteins derived from spines tissueand consider the efficiency, this method was not implemented. Purification of phospholipase A2 from spines venom of Ambon-Maluku Acanthasterplaci by using fractionated ammonium sulfate precipitation at 20% saturation of theheated crude venomwas done effectively. SDS-PAGE electrophoresis showed PLA2isolates has one protein band while the crude venom has four protein bands. PLA2isolates has a specific activity 20 times the specific activity of crude venom. Heatedthe crude venom at 60°C for 30 minutes followed by centrifugation for 30 minutes at15.000xg and 4°C separated PLA2 from the other heat sensitive proteins. This methodwas also implemented to purify PLA2 spines venom of Acanthaster planci fromSorong-Papua, but have not been successful. While PLA2 purification by usingethanol precipitation at a level of 80% saturation was not effective but increased thespecific activity into five times crude venom specific activity. This Experimentalresults were published in the International Journal of Pharma and Bio Science Vol 2 /issue 2 / Apr-June 2011 and the International Journal of Pharma and Bio Science 2012Oct; 3 (4) :( B) 603-608. Investigated of antibacterial activity by using disc diffusion method exhibited clearzone around the disc pre-added PLA2 on Staphylococcus aureus culture, indicatedPLA2 of Acanthaster planci spines venom has antibacterial activity againstStaphylococcus aureus. This experimental result was published in Internationaljournal of Pharma and Bio Sciene 2013 Apr; 4(2) : (B)1-5 Qualitative investigated of antiHIV activity by using PBMCs of HIV patient showed adecrease of the DNA protein band intensity in electrophoresis result of RT-PCR RNAsample of the HIV cultured treated with PLA2. Furthermore, quantitative analysis ofthe Green Fluorescence Particle results showed the decline significantly from 9.72%into 0.29% in the number of HIV-infected cells by PLA2 treatment, indicated PLA2of Acanthaster planci spines venom has antiHIV activity.This experimental result waspublished in Asian Pacific Journal of Tropical Medicine(2014) 412-420 The cost of PLA2 purification was paid for : 1) chemicals and equipmentconsumables, 2) electricity for the operation of the tools, 3) tools rental and 4) labor.The cost of PLA2 purification was Rp. 446.192,- per 50 grams spines with the resultsobtained was 4.622 mg PLA2. Miniscale purification costs performed in this studywas efficiently implemented which is the commercial prices PLA2 is ± 590,000rupiahs per mg (59,00 US dolar) (Worthington USA product of snake venomCrotalus amandetus PLA2). Thus purification of PLA2 from Acanthaster plancispines venom might be have a good prospect to be developed. Acanthaster planci survay was done on March 2013 in Eastern part Ambon water,especially in Liang (dusun Tanjung and dusun Batu Dua) and Pombo island obtainedthe average density value of 5.8 adult individuals per hectare. Satellite images (2006)downloaded from NASA website in June 2013 shown coral reefs area as the habitat ofAcanthaster planci is 94.83 acres. Total estimated of adult Acanthaster planci in thosearea was 550 and the availablelity number that can be used to produce PLA2 was 20%per month. , Spines venom of Acanthaster planci have various biological activities: lethal activity,hemolytic, myonecrotic, bleeding, increased capillary permeability, edema, phospholipase A2(PLA2), the activity of histamine release from mast cells and cardio vascular activity. Spinesvenom of Acanthaster planci containing phospholipase A2 (PLA2), plancitoxin which ishomologous with mammals deoxyribonuklease II and plancinin anticoagulant peptide.Previous studies prove that the venoms derived from animals contain variouscompounds that are potential to be developed as antibiotic and therapeutic agents to treat adisease. Acanthaster planci spines venom with various potential biological activity maycontribute in the medical field that can be input for the state revenue. Antimicrobial effectresults by hydrolysis activity of PLA2 on microbial cell membrane phospholipids can bebeneficial to the development of antibiotic agent. PLA2 purified from snake venom haveantibacterial activity against Staphylococcus aureus, Proteus vulgaris, Proteus mirabilis, andBurkholderia pseudomallei. In addition, PLA2 has antiHIV activity through inhibition of therelease mechanism of intracellular viral capsid proteins and assumed PLA2 blocking viralentry into host cells before the virus opens membranes and independently utilizekoreseptornya. PLA2 protect human T lymphocytes by blocking viruses that have outersheath containing phospholipids.Acanthaster planci is a predator threatens coral populations, especially when there is aoutbreak population. Acanthaster planci utilization for the production of PLA2 can be aneffort population control productively and make it more useful. Purification of Acanthasterplanci spines venom PLA2 has been done by Shiomi and colleagues by using a series ofchromatography columns which is relatively expensive and takes a few days, so a simple andfast method with a relatively low cost was developed in this study. The results of this studyare expected to be input for utilaization of Acanthaster planci to produce PLA2 that can bedeveloped as antibacterial and antiHIV agents.Experiments method were conducted in this study gave the following results: Venom extraction from the spines of Acanthaster planci was effective through theprocess of sonication at 20 kHz for 2x8 minutes (intensity 80% and 10 outputs).Venom was accumulated in extraction medium solution of 0.01 M phosphate bufferpH 7.0 containing 0.001 M CaCl2 called crude venom. Qualitative tested by usinghuman blood treated with crude venom (1: 1) showed the blood anticoagulation byplancinin contained in the venom, proves the extraction process successfully. At theprevious conducted on a method of extraction, the spines were blended first andfollowed by sonicated. To minimize contaminant proteins derived from spines tissueand consider the efficiency, this method was not implemented. Purification of phospholipase A2 from spines venom of Ambon-Maluku Acanthasterplaci by using fractionated ammonium sulfate precipitation at 20% saturation of theheated crude venomwas done effectively. SDS-PAGE electrophoresis showed PLA2isolates has one protein band while the crude venom has four protein bands. PLA2isolates has a specific activity 20 times the specific activity of crude venom. Heatedthe crude venom at 60°C for 30 minutes followed by centrifugation for 30 minutes at15.000xg and 4°C separated PLA2 from the other heat sensitive proteins. This methodwas also implemented to purify PLA2 spines venom of Acanthaster planci fromSorong-Papua, but have not been successful. While PLA2 purification by usingethanol precipitation at a level of 80% saturation was not effective but increased thespecific activity into five times crude venom specific activity. This Experimentalresults were published in the International Journal of Pharma and Bio Science Vol 2 /issue 2 / Apr-June 2011 and the International Journal of Pharma and Bio Science 2012Oct; 3 (4) :( B) 603-608. Investigated of antibacterial activity by using disc diffusion method exhibited clearzone around the disc pre-added PLA2 on Staphylococcus aureus culture, indicatedPLA2 of Acanthaster planci spines venom has antibacterial activity againstStaphylococcus aureus. This experimental result was published in Internationaljournal of Pharma and Bio Sciene 2013 Apr; 4(2) : (B)1-5 Qualitative investigated of antiHIV activity by using PBMCs of HIV patient showed adecrease of the DNA protein band intensity in electrophoresis result of RT-PCR RNAsample of the HIV cultured treated with PLA2. Furthermore, quantitative analysis ofthe Green Fluorescence Particle results showed the decline significantly from 9.72%into 0.29% in the number of HIV-infected cells by PLA2 treatment, indicated PLA2of Acanthaster planci spines venom has antiHIV activity.This experimental result waspublished in Asian Pacific Journal of Tropical Medicine(2014) 412-420 The cost of PLA2 purification was paid for : 1) chemicals and equipmentconsumables, 2) electricity for the operation of the tools, 3) tools rental and 4) labor.The cost of PLA2 purification was Rp. 446.192,- per 50 grams spines with the resultsobtained was 4.622 mg PLA2. Miniscale purification costs performed in this studywas efficiently implemented which is the commercial prices PLA2 is ± 590,000rupiahs per mg (59,00 US dolar) (Worthington USA product of snake venomCrotalus amandetus PLA2). Thus purification of PLA2 from Acanthaster plancispines venom might be have a good prospect to be developed. Acanthaster planci survay was done on March 2013 in Eastern part Ambon water,especially in Liang (dusun Tanjung and dusun Batu Dua) and Pombo island obtainedthe average density value of 5.8 adult individuals per hectare. Satellite images (2006)downloaded from NASA website in June 2013 shown coral reefs area as the habitat ofAcanthaster planci is 94.83 acres. Total estimated of adult Acanthaster planci in thosearea was 550 and the availablelity number that can be used to produce PLA2 was 20%per month. ]