STUDY OF APOPTOSIS INDUCTION BY Ulva fasciata AND Gracilaria verrucosa EXTRACTS

• Main Author: Fitria, Mona
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/34097
• Institution: Institut Teknologi Bandung

Seaweeds is one of Indonesia's natural resources which has an enormous development opportunities in the future. Indonesian seaweed production reached 10,2 million tons in 2014, but its uses only as food source such as vegetables, salad, and main source of agar production. It is one of many sources of natural bioactive compounds such as pigments, lipids, polysaccharides, proteins, phenolic compounds, flavonoids, terpenoids, steroids, glycosides and phytosterol. Those bioactive compounds can be utilized in the fields of pharmaceuticals and health, leading to the study of bioactivity of seaweed extracts as an antibacterial, antiviral, anti-inflammatory, antiproliferative and anticancer activities. However, research on utilization of the seaweed’s bioactive compounds from Indonesia is still very limited. Therefore, this research aims to study the ability to induce apoptosis of the extracts of Ulva fasciata and Gracilaria verrucosa harvested from Sayang Heulang, a beach in Pameungpeuk, Garut, West Java. The study begins with bioactive compounds extraction using chloroform p.a, methanol p.a and 50 mM phosphate buffer pH 7.6 with a ratio 2:1:0.8, resulting crude methanol-water and chloroform extracts. Ulva fasciata produced 35.67% (w/w) crude methanol-water extract and 0.80% (w/w) chloroform extract, while Gracilaria verrucosa produced 10.22% (w/w) crude methanol-water extract and 2.50% (w/w) chloroform extract. Mitochondria for apoptosis test were isolated from rat liver using buffer pH 7.4 containing 0.25 M sucrose, 10 mmol/l Tris, and 0.5 mmol/l EDTA. Apoptosis activity of the crude methanol-water and chloroform extracts was evaluated using mitochondrial visualization under light microscopy and the detection of cytochrome c released from mitochondria which were conducted spectrometrically by observing absorption at 550 nm. The treatment of 10% v/v crude methanol-water extract for five hours at 37 oC toward mitochondrial suspension leads to formation of mitochondrial agregates. Study of the released of cytochrome c showed that the treatment of 10% (v/v) crude methanol-water extract from Ulva fasciata released 5.68 µM cytochrome c which has equal to 15.1% of mitoxantrone 500 µM activity, while those of Gracilaria verrucosa showed 3.46 µM cytochrome c, which has equal to 9.2% of mitoxantrone 500 µM activity. Crude methanol-water extract that has positive effect to the apoptosis activity were then separated by column chromatography using Silica G-60 7733 and a stepwise elution of hexane acetone mixture (8:2, 5:5, 2:8 v/v) and methanol as eluent, resulting 10 fractions of those from Ulva fasciata and seven fractions of those from Gracilaria verrucosa. Further screening of those column chromatography fractions revealed that four apoptotic fractions were defined as U7, U8, U9, U10 from Ulva fasciata and G4, G5, G6, G7 from Gracilaria verrucosa. Study of the release of cytochrome c showed that the treatment of 20% (v/v) of those active fractions from Ulva fasciata, released 4.54 µM to 10.06 µM cytochrome c, while those of Gracilaria verrucosa released 3.94 µM to 7.90 µM cytochrome c. Amongs the four active fractions, U9 from Ulva fasciata showed highest activity, which induce to release 10.06 µM cytochrome c (which has equal to 26.7% of mitoxantrone 500 µM activity), while those of G6 from Gracilaria verrucosa released 7.90 µM cytochrome c (which has equal to 21.0% of mitoxantrone 500 µM activity). Further analysis of UV spectrum of those apoptotic induced fractions showed that U9 is a mixture of phenolic compounds, glycosides, and terpenoids, while those of G6 is a mixture of flavonoids, glycosides, and other phenolic compounds.