INTRODUCTION

As a maritime country Indonesia has abundant biodiversity including seaweeds or algae. Seaweeds contain various compounds as primary and secondary metabolites. Primary metabolites such as polysaccharides including alginat, carrageenan and agar, have been commercial used in food, nutraceutical, cosmetic and agricultural industries. However secondary metabolites are still less used. Therefore the economic value of brown seaweed is lower than red or green seaweed. So the aim of this study was to find the bioactivity of extract and fraction of brown seaweed.

Six species of brown seaweed (Sargassum ilicifolium, Sargassum binderi, Turbinaria decurrens, Turbinaria ornata, Padina australis and Hormophyta triquetra) has been studied the cytotoxicity activity to T47D and HeLa cell line. Turbinaria decurrens showed the cell growth inhibition for both cell lines. T. decurrens also showed the antioxidant activity and contain high fucoxanthin (^{Nursid et al., 2013}). Fucoxanthin is the main component of carotenoid nonprovitamin A, with C42-H58O6 formula (^{Kim et al., 2012}).

Fucoxanthin showed cytotoxic effects by inducing apoptosis of prostate cancer cell line PC-3 (^{Kotake-Nara et al., 2001}), leukemia HL-6 (^{Nakazawa et al., 2009}), and 6 colorectal cell lines, Caco-2, WiDr, HCT116, DLD-1, SW620, Colo205, Caco-2 cell lines (^{Takahashi et al., 2015}). Turbinaria decurrens Bory also contain fucoidan that has been studied showing neuroprotective (^{Meenakshi et al., 2016}) and hepatoprotective effects (Meenakshi et al., 2014).

Cancer is one of the leading causes of morbidity and mortality worldwide, 8.8 million deaths in 2015, colorectal cancer causes 774,000 deaths (WHO, 2017). Colon cancer can be prevented by selecting the appropriate foods and the lifestyle. The most effective treatment is surgical removal, and patients whose tumour cannot be removed are treated using chemotherapy and radiotherapy. Unfortunately, the five-year survival rate for metastatic colon cancer is below 10 % (^{Chen & Huang, 2009}). Therefore, this study used Turbinaria decurrens to study the activity to another cell line, that was colon cancer cell line HCT-116.

MATERIAL AND METHOD

Chemicals. Ethanol, n-hexane, ethyl acetate, aquadest, methanol, acetonitrile (Merck), Counting Cell Kit-8 (Dojindo Laboratories, Japan), 5-Fluorouracil (5-FU) (Kalbe), fucoxanthin (Sigma).

Brown seaweed. Brown seaweed Turbinaria decurrens was collected from Binuangen Beach, Banten, Indonesia. The collected seaweeds were washed with tap water and distilled water to remove salt and other debris then packed with iced to keep cool during the transportation to the laboratory.

Extraction and fractination. Two kg wet weight Fresh seaweed Turbinaria decurrens Bory were extracted with 70 % ethanol. Filtrate were evaporated in evaporator with vacuum. Crude extract was dissolved by aquadest (100 ml) and fractionated by liquid-liquid fractionation by n-hexane and ethyl acetate (each 100 ml x 3) respectively and both evaporated in vacuum to get n-hexane and ethyl acetate fractions. Ethanol-aqueous residue was evaporated to get ethanol fraction.

Analysis of fucoxanthin in the extract and fraction. Fucoxanthin content of the extract was analyzed using High Performance Liquid Chromatography (HPLC). Fucoxanthin in extract expressed as mg per 1 gram ethanolic extract and in fraction expressed as mg per 1 gram fraction. HPLC condition used the method as described by ^{Noviendri et al. (2011}). HPLC analysis carried out using a Shimadzu system equipped with a pump (LC-20AT), UV/Vis detector (SPD20A). Reversed-phase HPLC (RP-HPLC) analysis were carried out using a C18 RP column, 5 µm particle size, 250 mm x 4,6 mm (YMC Co., LTD). Fucoxanthin content was determined with methanol-acetonitrile (7:3, v/v) as the mobile phase at a flow rate 0.5 ml/min. The amount of fucoxanthin was quantified from the peak area using a standard curve with fucoxanthin standard (Sigma).

Cytotoxic assay. A Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Japan) were used to measure the cytotoxicity of ethanolic extract and n-hexane, ethyl acetate and ethanolic fraction of T. decurrens on human colon cancer cell HCT-116. 100 mL HCT-116 cell line (1000 cell/well) were grown in 96-well plates and preincubated for 24 h (temperature at 37 ºC, 5 % CO2). After 24 h, treated with 100 mL extract and fraction with various concentrations (0240 mg/mL). 5-fluorourasil (5-FU) was used as positive control and distributed with various concentrations 0-625 mg/mL (^{Davoodi et al., 2013}) and fucoxanthin standard (Sigma) as marker substance of brown seaweeds with various concentrations 0 - 150 mg/mL. All samples then incubated for 48 h in incubator. The extent of cell growth was assessed using a CCK-8 assay, CCK-8 solution (10 mL) was added to each well, followed by incubation for 1 h at 37 ºC. The absorbance at 450 nm was determined by multiplate reader. The inhibition percentage of cell growth were calculated with formula: (A-D)-(B-C)/(A-D) x 100 %, where A = control cell absorbance, B = compounds absorbance, C = controls compound absorbance, and D = control media absorbance. The inhibition concentration 50 (IC50) value is defined as the concentration of compound which inhibited 50 % of the cell growth (Nursid et al.).

RESULTS AND DISCUSSION

Fucoxanthin content. Fucoxanthin is main component of non-provitamin A carotenoid and has free radical scavenging activity due to unusual allenic double bonds (C=C=C) that is believed to be responsible for its higher antioxidant activity (^{Agatonovic-Kustrin et al., 2016}). Fucoxanthin has been investigated as antitumor and cancer-preventative function that indicate inducing G1 cell-cycle arrest and apoptosis in various cell lines (^{Satomi, 2017}).

Ethanol solvent was chosen for extraction because it provided the best fucoxanthin extraction yield from Phaeodactylum tricornutum (15.71 mg/g freeze-dried sample weight (Kim et al.). Fucoxanthin content was analyzed using HPLC in ethanolic extract, n-hexane, ethyl acetate and ethanolic fraction of Turbinaria decurrens. Based on retention time of fucoxanthin standard, peak of fucoxanthin detected in 7.8 min. Peak in thats retention time also showed in extract and fraction (Fig. 2a-d).

The results showed that of fucoxanthin content in the extract and fraction as shown in Table I were analysis using chromatogram of fucoxanthin standard. Fucoxanthin standard has retention time 7.905 min as shown in Figure 1, the extract and all fractions have similar chromatogram at the RT around 7.9 min as shown by red arrow in Figure 2. From this result the fucoxanthin concentration of the ethanolic extract, ethyl acetate fraction, n-hexane fraction, ethanolic fraction are 9.851 mg/g, 10.148 mg/g, 9.869 mg/g and 9.781 mg/g respectively.

Cytotoxicity test. Cytotoxic activity of the extract and fractions of T. decurrens was evaluated using CCK-8 assay. After 48 h of treatment, the mortality of HCT-116 cell was shown by treatment of extract, n-hexane and ethyl acetate fraction, but didn’t show inhibition activity from ethanolic fraction (Table II). IC50 values of extract and n-hexane, ethyl acetate fraction was 215 µg/ml, 1.512 µg/ml, 3.058 µg/ml, respectively.

Cytotoxic activity from T. decurrens to another cancer cell line has been reported. Methanolic extract showed cytotoxic activity to T47D, HepG2 and C26 cells with IC50 value were 172, 360, and 330 µg/ml . After fractionation of the Turbinaria decurrens Bory extract, the cytotoxicity increased with IC50 of n-hexane, ethyl acetate and methanol fraction were 43.1, 51.9, 383.0 µg/ml (Nursid et al.). This research indicated that extract T. decurrens was potential as an anticancer agent, n-hexane and ethyl acetate fraction also can be considered for the development of anticancer agent.

Cytotoxic activity of T. decurrens was compared with fucoxanthin standard and 5-fluorouracil as a positive control. Cytotoxic activity of extract was lower than fucoxanthin and 5-FU, while n-hexane and etyl acetate fraction seem higher than 5-FU but still lower than fucoxanthin.

Fucoxanthin showed inhibition values (IC50) of HCT-116 cell at 1.207 µg/ml, which means fucoxanthin is a potential anticancer agent and supported by antioxidant activity that has IC50 values at 1.0974 ppm (^{Zailanie et al., 2015}). Antiproliferative effect of cancer cell line also has been evaluated by ^{Asai et al. (2003}) and IC50 values on the proliferation of PC-3 cells was 4,6 µM for fucoxanthin. IC50 -of 5-FU was 11.843 µg/ml, its value was not significantly different from Davoodi et al., that showed IC50 of 5-FU after 48 h treatment to HCT-116 was 10 µg/ml.

From cytotoxic test, it’s seem not only fucoxanthin that affected the cytotoxic activity because it’s not showed in ethanolic fraction although it has fucoxanthin. Cytotoxic activity in brown seaweed was not always related to fucoxanthin because brown seaweed has another component, such as steroid, poysaccharide and phenol. 3-keto-22-epi28-nor-cathasteron and kolest-4-ene-3,6-dion has cytotoxic activity to HEPG-2 and HCT 116. Fucosterol also showed cytotoxic activity to HT-29, Caco-2 dan T47D (^{Hussain et al., 2016}). Fucoidan, a polysaccharide, has cytotoxic activity to HCT-15 with IC50 value was 75 µg/mL (^{Somasundaram et al., 2016}). Flavonoid that isolated from Turbinaria ornata showed antiproliferation to A549, PC-3, HCT-15 dan MG63 cells. Bromophenol had IC50 value at 10 µg/mL to HT1080 and HCT-8 cells (^{Kim, 2011}).

Fig. 1 Chromatograms of 25 ug/ml fucoxanthin standard. 

Fig. 2 Chromatograms of fucoxanthin in ethanolic extract (a), ethyl acetate (b), n-hexane (c), and ethanolic (d) fraction of T. decurrens. *red arrow indicated fucoxanthin. 

Table I Yield percentage and fucoxanthin content of T. decurrens extract and fractions. 

Table II Cytotoxicity against HCT-116 cell line. 

CONCLUSION

Extract, n-hexane and ethyl acetat fraction of Turbinaria decurrens Bory had cytotoxic activity against HCT-116 cell line, and contain fucoxanthin in extract and fraction.