Bioactive Profiling of Sargassum cristaefolium Lipid-Soluble Extract and Fraction from Indonesia with Their In-vitro Anti-Inflammatory and Antioxidant Activity Evaluation

Abstract

Sargassum is a popular brown seaweed genus which has been traditionally utilized by East Asian community as food and medicine. This brown seaweed is reported to exhibit several biological activities which promote human health, including anti-inflammatory and antioxidant activity. This research aimed to evaluate the potency of Indonesian S. cristaefolium as a source of antiinflammatory and antioxidant agents. To achieve the aforementioned general objective, this research was carried out through three main stages, i.e. 1) preliminary chemical characterization of brown seaweed obtained from four different water areas in Java - Indonesia, 2) preliminary screening of anti-inflammatory and antioxidant activity of brown seaweed crude lipid extracts, and 3) antiinflammatory and antioxidant evaluation of lipid-soluble fractions derived from selected brown seaweed sample and putative identification of bioactive compounds using ultra-high-performance liquid chromatography electrospray ionization orbitrap tandem mass spectrometry (UHPLC-ESI-ORBITRAP-MS/MS) instrument. Research was conducted at Chemistry Laboratory of Southeast Asian Food and Agricultural Science and Technology (SEAFAST) Center, Primate Research Center, and Tropical Biopharmaca Research Center of IPB University, Fat and Oil Laboratory, Food Science and Technology Faculty – University Putra Malaysia (UPM), and Institute of Bioscience of UPM. Results of preliminary chemical characterization of brown seaweeds obtained from different coastal areas showed that samples from southern waters of Java (Sayang Heulang Beach/SHB and Ujung Genteng Beach/UGB) were characterized by high contents of ash, alginate and fucose-containing sulfated polysaccharides (FCSPs) yield, and FCSPs’ fucose content, while samples from northern waters (Awur Bay/AB and Pari Island/PI) had a high amount of total sugar and crude fiber. Principle component analysis (PCA) result on lipid-soluble components showed different tendency that SHB and AB samples were located at close proximity and characterized by larger blade size, higher content of chlorophyll, fucoxanthin, carotenoid, PUFA, total n-3 fatty acids, total n-6 fatty acids, and also lower ratio of n-6 to n-3. Results of second research stage showed that treatment of RAW 264.7 macrophage cells with UGB and AB crude lipid extracts (12.5-50 μg/ml) significantly suppressed the nitric oxide production after lipopolysaccharide stimulation, both in pre-incubated and co-incubated cell culture model. Both crude lipid extracts showed strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and high ferric reducing antioxidant power (FRAP), which were amounted to 36.93-37.87 μmol trolox equivalent/g lipid extract and 681.58-969.81 μmol FeSO4/g lipid extract, respectively. AB samples was chosen for further research stage because this sample yielded the highest total lipid content with a selective anti-inflammatory effect and strong antioxidant activity. In the third stage, selected crude lipid extract was fractionated by normal-phase column chromatography, yielding three different lipid-soluble fractions, namely DCM (dichloromethane), ACT (acetone), and MeOH (methanol). The ACT fraction exerted strongest anti-inflammatory activity in both pre-incubated and co-incubated cell culture models. This was in accordance with its DPPH radical scavenging activity and its FRAP value. The anti-inflammatory effect of brown seaweed lipid-soluble fractions was most marked in the pre-incubated cell culture model. Metabolite profiling of lipid fractions was run by UHPLC-ESI-ORBITRAPMS/ MS. Orthogonal Projection to Latent Structures (OPLS) analysis was conducted to determine some features which were significantly correlated with the bioactivity of active fraction. There were 14 feature candidates considered from both positive and negative ionization mode datasets. Seven out of them were putatively identified as pheophytin a (1), all-trans fucoxanthin (2), 132-hydroxypheophytin a (3), pheophorbide a (4), 1-hexadecanoyl-2-(9Z-octadecenoyl)-3-O-β- D-galactosyl-sn-glycerol (6), 1-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-2- (9Z,12Z,15Z-octadecatrienoyl)-3-O-β-D-galactosyl-sn-glycerol (10), and 1- (9Z,12Z,15Z-octadecatrienoyl)-2-(6Z,9Z,12Z,15Z-octadecatetraenoyl)-3-O-β-Dgalactosyl- sn glycerol (12).

Sargassum adalah genus rumput laut cokelat yang populer dan telah digunakan secara tradisional oleh masyarakat Asia Timur sebagai pangan dan obat. Rumput laut cokelat ini dilaporkan menunjukkan berbagai aktivitas biologis yang bermanfaat bagi kesehatan, diantaranya adalah aktivitas anti-inflamasi dan antioksidan. Penelitian ini secara umum bertujuan untuk mengevaluasi potensi S. cristaefolium asal Indonesia sebagai agen anti-inflamasi dan antioksidan. Untuk mencapai tujuan umum tersebut, studi ini dilakukan melalui tiga tahapan utama, yaitu: 1) studi pendahuluan dalam karakterisasi profil kimia rumput laut cokelat dari empat wilayah perairan berbeda di Pulau Jawa - Indonesia, 2) studi penapisan aktivitas anti-inflamasi dan antioksidan dari ekstrak larut lemak/ekstrak lipid kasar rumput laut cokelat, dan 3) evaluasi aktivitas anti-inflamasi dan antioksidan dari fraksi larut lemak rumput laut cokelat dan identifikasi komponen bioaktif menggunakan instrumen ultra-high-performance liquid chromatography electrospray ionization orbitrap tandem mass spectrometry (UHPLC-ESI-ORBITRAP-MS/MS). Penelitian ini dilakukan di Laboratorium Kimia, Pusat Pengembangan Ilmu Pengetahuan dan Teknologi Pertanian dan Pangan Asia Tenggara, Pusat Studi Satwa Primata, dan Pusat Studi Biofarmaka - IPB University, Laboratorium Minyak dan Lemak, Fakultas Ilmu dan Teknologi Makanan – Universiti Putra Malaysia (UPM), dan Institut Biosains - UPM. Hasil studi pendahuluan karakterisasi profil kimia rumput laut cokelat yang diperoleh dari empat wilayah perairan berbeda menunjukkan bahwa sampel dari perairan selatan Jawa (Pantai Sayang Heulang/SHB and Pantai Ujung Genteng/UGB) dicirikan oleh tingginya kandungan abu/mineral, tingginya rendemen alginat dan fukoidan/fucose-containing sulfated polysaccharides (FCSPs), dan tingginya kandungan fukosa pada fraksi FCSPs. Adapun sampel dari perairan utara Jawa (Teluk Awur/AB dan Pulau Pari/PI) memiliki kandungan total gula dan total serat kasar yang tinggi. Hasil analisis komponen utama pada profil komponen larut lemak menunjukkan kecenderungan berbeda, dimana sampel SHB dan AB cenderung mengelompok berdekatan dan kedua sampel tersebut dicirikan oleh ukuran organ menyerupai daun/blade yang lebih lebar, tingginya kandungan klorofil, fukosantin, karotenoid, asam lemak tak jenuh jamak, asam lemak n-3, asam lemak n-6, dan rendahnya rasio asam lemak n-6:n-3. Hasil studi tahap kedua menunjukkan bahwa pemberian ekstrak UGB dan AB (12.5-50 µg/ml) pada sel makrofag RAW 264.7 dapat menekan secara signifikan produksi nitrit oksida yang diinduksi oleh lipopolisakarida, baik pada model pre-inkubasi maupun model ko-inkubasi. Kedua ekstrak lipid tersebut juga menunjukkan aktivitas peredaman radikal 2,2-difenil-1-pikrilhidrazil (DPPH) dan reduksi ion ferri yang kuat, yaitu masing-masing berkisar 36,93-37,87 µmol trolox ekuivalen/g ektrak lipid dan 681,58-969,81 µmol FeSO4/g ekstrak lipid. Sampel AB dipilih untuk digunakan pada tahap selanjutnya karena sampel tersebut menghasilkan rendemen lipid paling tinggi dengan efek anti-inflamasi yang selektif serta aktivitas antioksidan yang cukup kuat. Pada tahap ketiga, ekstrak lipid kasar terpilih kemudian difraksinasi menggunakan kolom kromatografi fase normal, sehingga dihasilkan tiga fraksi larut lemak berbeda, yaitu DCM (diklorometana), ACT (aseton), dan MeOH (metanol). Fraksi ACT menunjukkan aktivitas anti-inflamasi paling kuat pada kedua model studi anti-inflamasi yang diujikan. Hal ini seiring dengan tingginya aktivitas peredaman radikal DPPH dan kemampuan reduksi ion ferri. Efek anti-inflamasi dari fraksi larut lemak yang diujikan terlihat lebih besar pada model pre-inkubasi. Analisis profil metabolit dari tiga fraksi larut lemak dilakukan menggunakan UHPLC-ESI-ORBITRAP-MS/MS. Analisis orthogonal projection to latent structures (OPLS) dilakukan untuk menentukan beberapa kandidat metabolit yang diduga berkorelasi positif dengan aktivitas anti-inflamasi. Terdapat 14 kandidat fitur yang dihasilkan dari set data mode ionisasi positif dan negatif. Tujuh diantaranya telah berhasil diidentifikasi secara putatif dan terdiri atas feofitin a (1), all-trans fukosanin (2), 132-hidroksi-feofitin a (3), feoforbid a (4), 1-heksadekanoil-2-(9Z-oktadekenoil)-3-O-β-D-galaktosil-sn-gliserol (6), 1-(5Z,8Z,11Z,14Z,17Z-eikosapentaenoil)-2- (9Z,12Z,15Z-oktadekatrienoil) – 3 – O – β – D – galaktosil – sn – gliserol (10), dan 1-(9Z,12Z,15Z-oktadekatrienoil)-2-(6Z,9Z,12Z,15Z-oktadekatetraenoil)-3-O-β-D-galaktosil-sn gliserol (12).