Aktivitas Inhibisi Alpha-Amilase Hidrolisat Protein Alga Merah Gracilaria sp. secara In Vitro

Nisrina Salsabila, Dr. Tri Rini Nuringtyas, S.Si., M.Sc.

2023 | Skripsi | BIOLOGI

Pengobatan diabetes mellitus (DM) yang umum diadministrasikan adalah senyawa insulin dan non-insulin masih memiliki risiko efek samping tinggi. Ekstrak alga merah Gracilaria sp. telah diteliti memiliki efek inhibisi alpha-amilase (alpha-A) yang dilaporkan menjadi alternatif pengobatan DM yang lebih aman. Namun, penggunaan Gracilaria sp. hanya terbatas pada metabolit sekundernya dan belum dimanfaatkan hidrolisat proteinnya, padahal Gracilaria sp. dilaporkan memiliki kandungan total protein yang lebih tinggi dibandingkan Ulva sp. dan Sargassum sp. Penelitian ini dilakukan untuk mengamati kandungan protein total serta profil protein pada Gracilaria sp. dan menganalisis aktivitas inhibisi alpha-A yang diperoleh dari hidrolisat protein Gracilaria sp. Metode yang dilakukan adalah koleksi sampel Gracilaria sp. di Pantai Krakal, DI Yogyakarta, preparasi sampel dan uji proksimat, teknik isolasi protein dengan ammonium sulfat (AS) dengan kejenuhan 75%, 80%, 85%, dan 90% yang dikombinasikan dengan etanol 100%, analisis profil protein SDS-PAGE dengan pewarnaan CBB dan silver-stain, pengamatan aktivitas inhibisi enzim alpha-A dengan hidrolisat protein oleh hidrolisis tripsin pada rasio enzim:substrat (b/b): 1:40, 1:50, dan 1:60, serta analisis statistik dengan one-way ANOVA dan uji DMRT dengan tingkat kepercayaan 95%, di mana p kurang dari 0.05  adalah siginifikan. Hasil menunjukkan isolasi terbaik pada presipitasi AS 85 persen dengan rendemen protein 43,67%, kemudian profil protein dengan pita tertebal pada pewarnaan CBB berada pada ukuran kurang dari 17 kDa dan sekitar 25 kDa, sedangkan pada pewarnaan silver-stain didapatkan pita yang tebal pada ukuran rentang 9, 11, 20, dan 48 kDa. Sedangkan aktivitas inhibisi alpha-A terbaik ada pada hidrolisat protein 1:60, walaupun secara signifikan lebih rendah dari akarbosa pada 1000 ppm. Penelitian dapat dilanjutkan dengan perbedaan teknik presipitasi protein, purifikasi protein lebih lanjut, dan pengukuran inhibisi alpha-A dengan enzim protease yang berbeda.

Treatment of Diabetes mellitus (DM) that is commonly applied is insulin and non-insulin compounds which still have a high risk of side effects. Gracilaria sp. extract has been studied to inhibit the enzyme alpha-amylase (alpha-A), as considered safer drug target of DM. Even though it has a high protein content, the use of Gracilaria sp. is only limited to its secondary metabolites and its protein hydrolysate has not been utilized. Therefore, this research was conducted to analyze the best protein extraction method, the protein profile on SDS-PAGE, as well as to investigate alpha-A inhibitory activity on protein hydrolysates. The method used is the collection of samples of Gracilaria sp. at Krakal Beach, D I Yogyakarta, sample preparation and proximate analysis, ammonium sulfate protein precipitation with saturation of 75%, 80%, 85%, and 90% along with ethanol precipitation, protein profiling on CBB-stained and silver-stained SDS-PAGE, proceeded to analysis of alpha-A inhibitory activity from tryptic protein hydrolysis with enzyme:substrate: 1:40, 1:50, and 1:60 and last, statistical analysis using one-way ANOVA and DMRT test with 95% confidence level, where p less than 0,05 is significant. The results showed that the best protein isolate resulted from protein isolation with 85% saturated AS with a protein yield of 43,67%. Then, protein profiles can be observed on the thickest band appeared at less than 17 and approximately 25 KDa on CBB staining while sizes of approximately 9, 11, 20, and 48 kDa on silver-staining. The highest alpha-amylase inhibition activity was observed in the E:S treatment, namely 1:60, but significantly lower than the positive control of acarbose at 1000 ppm. Research can be continued with different protein precipitation techniques, further protein purification, and measurement of alpha-A inhibition with different protease enzymes.

Kata Kunci : Alpha-amilase, Ammonium Sulfat, Diabetes Mellitus, Gracilaria sp., Hidrolisat Protein