Hafiludin, - and Prof. Dr. Ir. Sri Andayani,, MS and Dr. Ir. Hartati Kartikaningsih,, M.Si and Dr. Ir. Muhamad Firdaus,, MP (2021) Pemanfaatan Ekstrak Siput Laut Paromoionchis tumidus sebagai Antiinflamasi pada Limfosit Mencit yang Diinduksi Lipopolisakarida Melalui Inhibisi Jalur TLR4 dan NF-kB. Doctor thesis, Universitas Brawijaya.
Abstract
Sumberdaya laut belum termanfaatkan secara optimal dalam berbagai bidang terutama sebagai sumber senyawa bioaktif dalam bidang pangan dan obat-obatan. Kekayaan sumber kimia dan biologis membangkitkan minat bagi para ilmuan dan peneliti ilmiah. Organisme yang memproduksi produk alam laut dibagi menjadi tiga kelas utama biologi: mikroorganisme laut (termasuk fitoplankton), alga/ganggang laut dan invertebrata laut. Siput laut Paromoionchis tumidus merupakan hewan invertebrata laut yang memiliki kemampuan pertahanan diri terhadap perubahan lingkungan dan predator berupa sekresi dari kulitnya. Sekresi kulit tersebut mengandung protein dan asam amino serta taurin yang bisa diaplikasikan sebagai kandidat antiinflamasi. Penggunaan obat dan agen biologis antiinflamasi sampai saat ini masih memberikan efek samping terhadap kesehatan. Pencarian bahan aktif dari lautan sebagai kandidat antiinflamasi masih diperlukan. Penelitian komprehensif tentang ekstrak siput laut dan manfaatnya sebagai antinflamasi masih perlu dikembangkan. Tujuan penelitian ini adalah (1), Menganalisis karakteristik siput laut Paromoionchis tumidus; (2) Menganalisis screening senyawa bioaktif dan probabilitas antiinflamasi peptida dan taurin ekstrak siput laut Paromoionchis tumidus secara bioinformatik; (3) Menganalisis pengaruh pemberian ekstrak siput laut Paromoionchis tumidus terhadap inflamasi sel limfosit mencit yang diinduksi lipopolisakarida secara in vitro; (4) Menganalisis pengaruh pemberian ekstrak siput laut Paromoionchis tumidus terhadap inflamasi sel limfosit mencit yang diinduksi lipopolisakarida secara in vivo. Penelitian ini dibagi dalam 4 tahap yaitu: (1) Ekstraksi dan karakterisasi siput laut Paromoionchis tumidus; analisis yang dilakukan pada tahap ini yaitu: analisis proksimat, analisis asam amino, analisis berat molekul protein, analisis kandungan taurin, dan aktivitas antioksidan ekstrak siput laut. (2) Screening senyawa bioaktif dan probalititas antiinflamasi ekstrak siput laut Paromoionchis tumidus secara bioinformatik; analisis yang dilakukan pada tahap ini yaitu: identifikasi senyawa ekstrak siput laut, screening senyawa bioaktif pepptida dan taurin ekstrak siput laut, probabilitas antiinflamasi ekstrak siput laut, dan interaksi senyawa dalam ekstrak siput laut dengan docking. (3) Pengujian antiinflamasi ekstrak kasar taurin siput laut Paromoionchis tumidus secara in vitro; analisis yang dilakukan pada tahap ini yaitu: isolasi sel limfosit mencit, kultur sel limfosit, pewarnaan intraseluler dan ekstraseluler, analisis flowcytometry untuk melihat ekspresi sitokin. (4) Pengujian antiinflamasi ekstrak kasar taurin siput laut Paromoionchis tumidus secara in vivo; analisis yang dilakukan pada tahap ini yaitu: pemeliharaan hewan uji mencit, perlakuan induksi ekstrak kasar taurin dan lipopolisakarida, isolasi sel limfosit, pewarnaan intraseluler dan ekstraseluler, analisa flowcytometry, dan histologi jaringan hewan uji. Siput laut Paromoionchis tumidus kaya akan protein yaitu sebesar 9,32% (basah) atau 60,82% (kering) dengan berat molekul berkisar 22,53-49,61 kDa. Kandungan total asam amino 6,14% (basah) dan 53,87% (kering). Siput laut dalam xi bentuk segar mempunyai kandungan taurin berkisar antara 53,568-158,784 μg/mL. Siput laut dalam bentuk ekstrak mempunyai kandungan taurin berkisar antara 155,58-229,87 μg/mL. Ekstrak siput laut Paromoionchis tumidus mempunyai berat molekul antara 31,1-117,6 kDa dengan intensitas terbesar terdapat pada 31 dan 37 kDa. Aktivitas antioksidan DPPH pada ekstrak berkisar antara 27,10-140,20 μg/mL dan aktivitas terbaik terdapat pada bagian mucus. Ekstrak siput laut mengandung senyawa peptida berupa L-Glutamic acid, Proline, DL-β-Leucine, N-Acetyl-L-leucine, Valylproline, Isoleucine, L-Phenylalanine, Leucylproline, Cyclo(leucylprolyline), serta senyawa taurin. Senyawa komplek peptida dan taurin ekstrak siput secara bioinformatik berpotensi sebagai agen antiinflamasi dan dapat mengikat protein inflamasi berupa NF-kB, TNF-α, IL-6, IFN-γ dan IL-10. Ekstrak siput laut Paromoionchis tumidus dapat mempengaruhi inflamasi sel limfosit mencit yang diinduksi lipolisakarida secara in vitro. Ekstrak kasar taurin siput laut dapat menghambat aktivasi sel T dan sel B, menghambat jalur pathway TLR4 dan NF-kB, menekan ekspresi sitokin proinflamasi dan meningkatkan ekspresi sitokin antiinflamasi. Ekstrak kasar taurin bagian mucus 250 ppm merupakan perlakuan terbaik dalam menekan aktivasi sel T CD4 sebesar 10,85±0,98%, sel T CD8 sebesar 4,83±1,07% dan sel B B220 sebesar 41,98±4,19%. Ekstrak siput laut dapat menekan aktivasi sel T naïve CD4 terbaik pada perlakuan bagian utuh 500 ppm sebesar 33,07±1,03%, sedangkan penekanan aktivasi sel T naïve CD8 pada perlakuan daging 500 ppm sebesar 29,35±2,49%. Ekstak bagian mucus 500 ppm memberikan penekanan terbaik terhadap ekspresi sinyal TLR4 sebesar 0,48±0,20% dan ekspresi sinyal transduksi NF-kB sebesar 0,45±0,26%. Ekstrak daging 500 ppm menekan ekspresi sinyal sitokin proinflamasi TNF-α sebesar 0,02±0,02%, sedangkan ekpresi sitokin IFN-γ pada perlakuan mucus 250 ppm sebesar 0,54±0,13%. Ekstrak siput laut dapat meningkatkan ekspresi sitokin antiinflamasi IL-10 pada perlakuan mucus 500 ppm sebesar 1,24±0,25%. Ekstrak siput laut Paromoionchis tumidus dapat mempengaruhi inflamasi sel limfosit mencit yang diinduksi lipopolisakarida secara in vivo. Pemberian ekstrak siput laut dapat menekan aktivasi sel T naïve CD4 terbaik sebesar 56,63± 0,01% pada konsentrasi 1000 ppm. Ekstrak siput laut dapat menekan pengaktivan sel T naïve CD8 sebesar 84,91±0,01% pada konsentrasi 1000 ppm. Ekstrak siput laut dapat menekan ekspresi reseptor TLR3 pada konsentrasi 1000 ppm sebesar 6,03±0,00%. Pemberian ektrak siput laut dapat menekan ekspresi sitokin proinflamasi TNF-α sebesar 2,98±0,01% pada konsentrasi 500 ppm dan sitokin IL-6 pada konsentrasi tinggi 1000 ppm sebesar 9,034±0,01%. Ekstrak siput laut dapat meningkatkan ekspresi sitokin antiinflamasi IL-10 sebesar 7,39±0,01% pada konsentrasi 1000 ppm. Ekstrak siput laut dapat menurunkan kadar MDA limfosit sebesar 6,27±0,21%. Penggunaan ekstrak siput laut dapat mencegah kerusakan sel mucosa jejenum usus dengan rata-rata panjang antara 141,118-362,25 μm. Kesimpulan hasil penelitian bahwa siput laut Paromoionchis tumidus kaya akan kandungan protein dan taurin. Ekstrak siput laut dalam bentuk peptida dan taurin berpotensi sebagai agen antiinflamasi melalui inhibisi jalur TLR dan NF-kB pada mencit inflamasi yang diinduksi dengan LPS.
English Abstract
Marine resources provide pivotal roles as a tremendous source of bioactive compounds for medicines and foods; unfortunately, they are still underutilized. To fill the gap, numerous scientists and researchers around the world have been actively motivated to scientifically investigate the resources for further use. As well known, there are three major biological classes of marine organisms producing marine products: marine microorganisms (including phytoplankton), algae and marine invertebrates. Sea slug Paromoionchis tumidus belongs to a marine invertebrate, possesses incredible defense mechanism using chemicals secreted by skin against environmental changes and predators. The chemical contains variety of amino acids and proteins such as taurine which is a considerable candidate of anti-inflammatory agent. The use of anti-inflammatory drugs and biological agents still has side effects on health. The search for natural products from the oceans as anti-inflammatory candidates is still needed. Future researches on taurine isolated from sea slug are required, mainly regarding its anti-inflammatory properties. The aim of this work is (1) to analyze the characteristic of sea slug Paromoionchis tumidus; (2) screening of bioactive compounds and the probability of anti-inflammatory peptides and taurines from Paromoionchis tumidus sea slug extract by bioinformatics; and analyze the effects of taurine crude extracts on inflammatory responses in mice lymphocyte cells induced with lipopolysaccharide via (3) in vitro and (4) in vitro experiments. The experiments were divided into 4 stages. First, we have extracted and characterized sea slug, quantified taurine and chemical content in sea slug, including proximate, amino acids, molecular weight of protein, taurine present in extract, and antioxidant activity. Second, screening of bioactive compunds and probability anti-inflammmatory activity in extract by bioinformatics was characterized, covering, indentification bioactive compunds and docking of anti-inflammatory of peptides and taurine . Third, sea slug extract was tested in vitro for its anti-inflammatory activity with the following steps: isolation of mice lymphocyte cells, the cell culture, intracellular and extracellular staining, flowcytometry analysis for evaluation of cytokine expression. Last, we confirmed the response of the sea slug extract against anti-inflammation via in vivo experiment as follows: mice preparation and maintenance, administration of the extracts to the mice and induction by lipopolysaccharide, isolation of lymphocyte cells, intracellular and extracellular staining, flowcytometry analysis, and histological studies. As the results, sea slug Paromoionchis tumidus showed the rich source of protein, reaching up to 9.32% (fresh sample) and 60.82% (dried sample) with molecular weight of 22.53-49.61 kDa. The total of amino acid in fresh and dried sample was 6.14% and 53.87%, respectively. The taurine in fresh sea slug reached 53.568-158.784 μg/mL. In the form of extract, content of taurine ranged from 155.58 to 229.87 μg/mL. Molecular weight of the extract was recorded between 31.1-117.6 kDa with the highest intensity at 31 and 37 kDa. Using DPPH assay, the extract demonstrated antioxidant activity of 27.10-140.20 μg/mL. Moreover, the sea slug extract from mucus showed the best, containing peptides such as L-Glutamic acid, Proline, DL-β-Leucine, N-Acetyl-L-leucine, Valylproline, Isoleucine, L-Phenylalanine, Leucylproline, Cyclo(leucylprolyline), and taurine compound. Furthermore, we found complex compunds of peptides and taurine in sea slug extract, it’s have the potential as anti-inflammatory agents by bioinformatics and can bind the protein of anti-inflammatory such as NF-kB, TNF-α, IL-6, IFN-γ and IL-10. Extract from sea slug Paromoionchis tumidus incredibly affected inflammation of mice lymphocyte cells induced by lipopolysaccharide via in vivo experiment. It enabled to suppress activity of T-cells and B-cells, hamper TLR4 and NF-kB pathway, reduce cytokine expression, but raise production of anti-inflammatory cytokine. Further experiment revealed that sea slug extract from mucus at dose of 250 ppm showed the most powerful treatment on reducing activity of T-cell CD4 (10.85±0.98%), T-cell CD8 (4.83±1.07%) and B-cell B220 (41.98±4.19%). Additionally, the extract could attenuate activation of T naïve CD4 (up to 33.07±1.03%), observed in dose of 500 ppm, meanwhile, activation of T-cell naïve CD8 was best suppressed at dose of 500 ppm, reducing reduction up to 29.35±2.49%. Extract from mucus at dose of 500 ppm resulted in the strongest effect on inhibition of TLR4 signal expression up to 0.48±0.20% and expression of NF-kB signal transduction up to 0.45±0.26%. In addition, extract from muscle at dose of 500 ppm was able to suppress expression of proinflammatory cytokine TNF-α reaching up to 0.02±0.02%, meanwhile the suppression of IFN-γ cytokine by mucus at dose of 250 ppm was 0.54±0.13%. In this regard, we could argued that sea slug extract enabled to the release of anti-inflammatory cytokine IL-10 up to 1.24±0.25% using dose of 500 ppm. Furthermore, extract from sea slug enabled to affect mice lymphocyte cells previously induced by lipopolysaccharide in in vivo experiment. The extract reduced activation of T-cell naïve CD4, up to 56.63± 0.01% at dose 1000 ppm, while at similar concentration, the extract could inhibit activation of T-cell naïve CD8, reaching up to 84.91±0.01%. Besides, the extract was evidenced able to lessen expression of TLR3 receptors up to 6.03±0.00% at concentration of 1000 ppm. We also highlighted the reduction of TNF-α pro-inflammatory cytokine up to 2.98±0.01% at dose of 500 ppm, and IL-6 cytokine up to 9.034±0.01% at 1000 pm. On the other hand, the extract induced production of anti-inflammatory cytokine IL-10 up to 7.39±0.01% at dose of 1000 ppm. The MDA was decresed after the administration of sea alug extract at dose 1000 ppm. Extract can prevent damage to intestinal jejenum mucosa cells with average length between 141.118-362.25 μm. To sum up, sea slug Paromoionchis tumidus was rich in protein and taurine active components, found in its variety of body parts, i.e. whole, mucus, viscera, and muscle. Sea slug extracts in the form peptides and taurine suggested its potential as anti-inflammatory agent via inhibitory TLR and NF-kB pathway on LPS-induced mice.
Item Type: | Thesis (Doctor) |
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Identification Number: | 0621080007 |
Subjects: | 600 Technology (Applied sciences) > 660 Chemical engineering and related technologies > 660.6 Biotechnology |
Divisions: | S2/S3 > Doktor Ilmu Perikanan dan Kelautan, Fakultas Perikanan dan Ilmu Kelautan |
Depositing User: | Budi Wahyono Wahyono |
Date Deposited: | 11 Jan 2022 07:18 |
Last Modified: | 01 Oct 2024 01:26 |
URI: | http://repository.ub.ac.id/id/eprint/188166 |
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