Ekstrak etanol Amphora sp. untuk meningkatkan kekebalan ikan Kerapu Cantang (Epinephelus fuscoguttatus x Epinephelus lanceolatus) terhadap infeksi Viral Nervous Necrosis

Khumaidi, Ach and Dr. Ir. Mohamad Fadjar,, M. Sc. and Feni Iranawati,, S.Pi., M.Si., Ph.D and Dr. Yuni Kilawati,, S.Pi., M.Si. (2020) Ekstrak etanol Amphora sp. untuk meningkatkan kekebalan ikan Kerapu Cantang (Epinephelus fuscoguttatus x Epinephelus lanceolatus) terhadap infeksi Viral Nervous Necrosis. Doctor thesis, Universitas Brawijaya.

Abstract

Sejak tahun 2006, ikan Kerapu hybrid “Cantang” (Epinephelus fuscoguttatus x Epinephelus lanceolatus) menjadi primadona baru bagi para pembudidaya ikan dengan nilai kelebihan pertumbuhan cepat, rasa dinilai lebih enak, lebih tahan terhadap penyakit, dan harga yang tinggi. Serangan penyakit yang disebabkan oleh virus ternyata tetap menjadi kendala terbesar bagi produksi ikan Kerapu Cantang. Viral nervous necrosis (VNN) termasuk salah satu virus mematikan yang dapat menyebabkan mortalitas Kerapu Cantang hingga 100%. Pemberian bioaktif dari bahan alami mikroalga dinilai dapat meningkatkan sistem imun ikan untuk menanggulangi serangan VNN. Amphora sp. memiliki berbagai macam bioaktif yang diharapkan mampu meningkatkan proliferasi sel darah merah (eritrosit), sel darah putih (leukosit), hemoglobin (Hb), dan hematokrit (Hc) guna meningkatkan sistem imun ikan Kerapu Cantang dalam menghadapi serangan VNN. Tujuan penelitian ini untuk memproduksi ekstrak kasar Amphora sp. dan melakukan pengkajian pemanfaatannya sebagai imunostimulan ikan Kerapu Cantang. Penelitian dilakukan dalam tiga tahap: 1) Produksi, metode ekstraksi, dan karakterisasi bioaktif Amphora sp. serta analisis potensi obat secara in-silico, 2) Penentuan konsentrasi ekstrak Amphora sp. pada ikan Kerapu Cantang dan perannya meningkatkan sistem imun Kerapu Cantang, 3) Penentuan konsentrasi ekstrak terbaik dalam peningkatan sintasan atau kelulushidupan Kerapu Cantang yang diinfeksi VNN. Penelitian pertama bertujuan mengkaji pengaruh metode ekstraksi terhadap kandungan bioaktif ekstrak Amphora sp. Isolat Amphora sp. yang digunakan pada penelitian ini diperoleh dari Laboratorium Pakan Alami BPBAP Situbondo. Penelitian dilakukan dengan dua tahapan yaitu: 1) melakukan pengujian penggunaan metode ekstraksi berbeda: sonikasi (SON), maserasi (MAS), dan gabungan sonikasi-maserasi (SON-MAS), dengan menggunakan pelarut etanol absolute, 2) melakukan analisis kandungan fitokimia (kualitatif dan kuantitatif), analisis aktivitas antioksidan, skrining bioaktif dengan LC-HRMS. Hasil penelitian menunjukkan metode SON-MAS menghasilkan rendemen tertinggi 9.8% dibandingkan dengan MAS 8.71% dan SON 8.47%. analisis fitokimia metode SON-MAS dan MAS memiliki kandungan senyawa flavonoid, alkaloid, dan triterpenoid, sedangkan metode MAS menunjukkan dua senyawa yaitu alkaloid dan triterpenoid. Analisis fitokimia secara kuantitatif kandungan fenol dan flavonoid menunjukkan metode SON memilki nilai yang lebih tinggi yaitu fenol 34.2 mg GAE/g dan flavonoid 15.2 mg QE/g. Aktivitas antioksidan dalam menghambat radikal bebas DPPH terbaik terlihat pada metode SON dengan nilai IC50 745.2 ppm. Skrining LC-HRMS menunjukan terdapat 6 senyawa dominan yaitu valine, L-Norleucine, stearamide, palmitoleic acid, isotretinoin, arachidonic acid. Penelitian kedua bertujuan mengkaji potensi obat secara in-silico yaitu absorption, distribution, metabolism, excretion, and toxicity (ADME/T) serta prediksi aktivitas biologis (Pass prediction). Hasil analisis ADME/T pada enam senyawa ekstrak Amphora sp. memiliki potensi obat yang baik berdasarkan analis rule of five dan toksisitas dengan software Protox II. Enam senyawa ekstrak Amphora sp. memiliki aktivitas biologis sebagai agen antiinflamasi, antifungi, antibakteri, antivirus, dan antineoplastik dengan kisaran nilai probability (pa) 0.260 – 0.873 melalui analisis Pass prediction. Penelitian ketiga bertujuan mengetahui pengaruh pemberian ekstrak Amphora sp. terhadap gejala klinis, profil darah dan histologi ikan Kerapu Cantang yang diuji tantang dengan VNN. Pada penelitian ini dilakuan lima perlakuan: kontrol (+) ikan Kerapu Cantang tanpa perlakuan ekstrak Amphora sp. dan diinfeksi dengan VNN, kontrol (-) ikan Kerapu Cantang tanpa perlakuan ekstrak Amphora sp. dan tanpa penginfeksian VNN, perlakuan A1 (pemberian ekstrak Amphora sp. konsentrasi 33 μg/ml dan diuji tantang dengan VNN), perlakuan A2 (pemberian ekstrak Amphora sp. konsentrasi 66 μg/ml dan diuji tantang dengan VNN), dan perlakuan A3 (pemberian ekstrak Amphora sp. konsentrasi 99 μg/ml dan diuji tantang dengan VNN). Pemberian ekstrak Amphora sp. dilakukan dengan cara disonde sebanyak 5 kali selama masa pemeliharan pada masing-masing perlakuan ekstrak yaitu pada hari ke-0, hari ke-5, hari ke-10, hari ke-20, dan hari ke-25 dengan dosis ekstrak 1 ml, dan uji tantang dilakukan pada hari ke-15 dengan dosis suspense VNN 0.1 ml (142385 salinan/μl) pada bagian intramuskular (IM). Pengamatan dilakukan pada profil darah (eritrosit, Hb, dan Hc, leukosit). Hasil penelitian menunjukkan perlakuan A3 memberikan respon imun terbaik dengan gejala klinis yang terlihat normal sampai akhir masa pemeliharaan dan profil darah: rerata eritrosit (2.710.000 sel/mm3), rerata Hb (8.5%), rerata Hc (33.7%), dan rerata leukosit (530.000 sel/mm3). Penelitian keempat bertujuan mengetahui tingkat sintasan ikan Kerapu Cantang yang beri ekstrak Amphora sp. dan diuji tantang dengan VNN. Pada penelitian dilakukan pengamatan tingkat mortalitas dan sintasan selama masa pemeliharaan (hari ke-30) pada masing-masing perlakuan. Pada penelitian ini, kontrol (+) memiliki nilai sintasan 0% (kematian 100%) dan kontrol (-) memiliki nilai sintasan 100% (kematian 0%). Perlakuan ekstrak Amphora sp. 99 μg/ml (A3) memberikan hasil terbaik dengan nilai sintasan 83.3%, dan diikuti 66 μg/ml (A2) dan 33 μg/ml (A1) masing-masing dengan nilai sintasan 36.7% dan 33.3%. Secara keseluruhan disimpulkan bahwa metode ekstraksi dengan metode sonikasi (SON) dinilai lebih efisien dalam menghasilkan bahan imunostimulan. Ekstrak Amphora sp. memiliki aktivitas sebagai imunostimulan dengan meningkatkan profil darah dan normalitas jaringan. Konsentrasi 99 μg/ml memberikan respon imun dan sintasan terbaik, serta dinilai efektif untuk meningkatkan tanggap kebal ikan Kerapu Cantang terhadap serangan VNN.

English Abstract

Since 2006, the "Cantang" hybrid grouper (Epinephelus fuscoguttatus x Epinephelus lanceolatus) has been the new favorite of fish farmers with added value, good taste, more resistance to disease and high prices. The disease attack which was caused by a virus turned out to be the biggest challenge for Cantang grouper fish production. Viral nervous necrosis (VNN) is one of the deadly viruses which can cause mortality of Cantang Grouper up to 100%. Giving bioactive from microalgae natural substances is considered to be able to increase the fish's immune system to cope with VNN attacks. Amphora sp. has a variety of bioactives which are expected to increase the proliferation of red blood cells (erythrocytes), white blood cells (leukocytes), hemoglobin (Hb), and hematocrit (Hc) in order to increase the immune system of Cantang grouper fish in the face of VNN attacks. The purpose of this study was to produce crude extracts of Amphora sp. and assessing its use as an immunostimulant for Cantang grouper fish. The study was conducted in four stages: 1) extraction methods and bioactive characterization of Amphora sp., 2) analysis of drug potential bioactive of Amphora sp. by in-silico method, 3) determination of the concentration of Amphora sp. in Cantang grouper fish and their role in enhancing Cantang grouper immune system, 4) determination of the best extract concentration in increasing survival of Cantang grouper infected by VNN. The first study aimed to examine the effect of the extraction method on the bioactive content of Amphora sp. Diatome Amphora sp. used in this study were obtained from the Natural Feed Laboratory, BPBAP Situbondo. The study was conducted in two stages: 1) testing the use of different extraction methods: sonication (SON), maceration (MAS), and combined sonication-maceration (SON-MAS), using absolute ethanol solvent, 2) conducting phytochemical content analysis (qualitative and quantitative), analysis of antioxidant activity, bioactive screening with LC-HRMS. The results showed the SON-MAS method yielded the highest yield of 9.8% compared to MAS 8.71% and SON 8.47%. Phytochemical analysis SON-MAS and MAS methods contain flavonoid, alkaloid, and triterpenoid compounds, while the MAS method shows two compounds namely alkaloid and triterpenoid. Quantitative phytochemical analysis of phenol and flavonoid content shows that SON method has a higher value, namely phenol 34.2 mg GAE/g and flavonoid 15.2 mg QE/g. The best antioxidant activity in inhibiting DPPH free radicals is seen in the SON method with an IC50 value of 745.2 ppm. LC-HRMS screening showed that there were 6 dominant compounds, namely valine, L-Norleucine, stearamide, palmitoleic acid, isotretinoin, arachidonic acid. The second study aims to examine the potential of drugs by in-silico method, namely absorption, distribution, metabolism, excretion, and toxicity (ADME/T) and prediction of biological activity (Pass prediction). The results of ADME/T analysis on six compound of Amphora sp. has good drug potential based on the rule of five analyst and toxicity with Protox II software. Six compound extracts of Amphora sp. has biological activity as an anti-inflammatory, antifungal, antibacterial, antiviral, and antineoplastic agent with a probability (pa) value range of 0.260 - 0.873 through Pass prediction analysis. The third study aimed to determine the effect of Amphora sp. on clinical symptoms, blood profile and histology of Cantang grouper fish that were tested challenged with VNN. In this study five treatments were carried out: control (+) of Cantang grouper fish without Amphora sp. extract and infected with VNN, control (-) Cantang grouper fish without extract Amphora sp. extract and without VNN infection, A1 treatment (administration extract of Amphora sp. concentration 33 μg/ml and tested challenged with VNN), A2 treatment (administration extract of Amphora sp. concentration 66 μg/ml and tested challenged with VNN), and A3 treatment (administration Amphora sp. extract concentration 99 μg/mL and tested challenged with VNN). Amphora sp. carried out by round as much as 5 times during the maintenance period on each extract treatment that is on the 0th day, 5th day, 10th day, 20th day, and 25th day with a dose of 1 ml extract, and The challenge test was carried out on the 15th day with a 0.1 ml VNN suspense dose (142385 copies/μl) on the intramuscular (IM) section. Observations were made on blood profiles (erythrocytes, Hb, and Hc, leukocytes). The results showed that A3 treatment gave the best immune response with clinical symptoms that looked normal until the end of the maintenance period and blood profile: mean erythrocytes (2,710,000 cells/mm3), average Hb (8.5%), average Hc (33.7%), and average leukocytes (530,000 cells/mm3). The fourth study aims to determine the level of survival of Cantang grouper fish which extract Amphora sp. and challenged with VNN. In the study, mortality and survival rates during the maintenance period (30th day) were observed in each treatment. In this study, control (+) had a survival rate of 0% (death 100%) and control (-) had a survival rate of 100% (death 0%). Amphora sp. 99 μg / mL (A3) gave the best results with a survival value of 83.3%, and followed by 66 μg/mL (A2) and 33 μg/mL (A1) respectively with a survival value of 36.7% and 33.3%. Overall, it was concluded that the extraction method with sonication (SON) method was considered more efficient in producing immunostimulant material. Amphora sp. has immunostimulant activity by increasing blood profile and tissue normality. The concentration of 99 μg/m provides the best immune and survival response, and is considered effective in increasing the response of the Cantang grouper fish to VNN attacks.

Item Type:	Thesis (Doctor)
Identification Number:	0620080006
Uncontrolled Keywords:	Amphora sp., hybrid grouper, sonication, immunostimulant, VNN.
Subjects:	600 Technology (Applied sciences) > 660 Chemical engineering and related technologies > 660.6 Biotechnology
Divisions:	S2/S3 > Magister Bioteknologi Perikanan dan Kelautan, Fakultas Perikanan dan Ilmu Kelautan
Depositing User:	Budi Wahyono Wahyono
Date Deposited:	11 Jan 2022 06:31
Last Modified:	17 Apr 2023 02:07
URI:	http://repository.ub.ac.id/id/eprint/188151

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