Aplikasi metabolomik untuk mengidentifikasi komponen bioaktif: komponen antibakteri dari ekstrak buah takokak (Solanum torvum Swart)

Abstract

Solanum torvum Swartz, commonly known as Takokak, has been traditionally used in Indonesia to cure several diseases. Fruits of Solanum torvum Swartz are used to cure anti-edemic, as analgesic, to improve blood circulation, to alleviate pain, as antitusive, as anti-inflammatory, to relieve stomach pain, toothache, cataracts, menstruation disorders, hemorrhoids, sore breasts, influenza, swelling, ulcers, soreness, sore waist, high uric acid, bone loss, heart palpitations, and for detoxification (Andarwulan et al. 2012). The antibacterial activity of Takokak fruit has been reported as well (Chah et al. 2000, Bari et al. 2010, Sivapriya et al. 2011). However, up to now no reports yet on compounds responsible for takokak’s antibacterial activity. Metabolomics has been found to be more efficient for identification tool of active principles in a complex mixture such as plant extract than the conventional way, which is known as bioassay guided isolation. To achieve this, a specific extraction method that is able to extract the widest possible range of metabolites, and a suitable analytical tool to detect them are required (Yuliana et al. 2011). From 4507,5 g fresh Takokak fruits 376 g dried powder was obtained. Extraction and fractionation with diverse polarity of solvents gave 5,62 g of methanol extract (M), 3,09 g of n-hexane fraction (H), 1,37 g of chloroform fraction (K), 2,59 g of ethyl acetate fraction (E), and 11,78 g of water fraction (A). The result of antibacterial activity indicated that among all the tested extracts and fractions, M extract and K fraction exhibited medium antibacterial activity against Staphylococcus aureus while others did not. Inhibition zone diameters of M extract were between 4-4,5 mm and K fraction were between 6,5–6,8 mm. The results of TLC showed 6 spots of M extract, 1 spot of H fraction, 10 spots of K fraction, 3 spots of E fraction, and 1 spot of A fraction. The TLC spots appearance (blue fluorescence in UV 366 and 254 nm) suggested that steroid components are probably the active components of Takokak fuit. Furthermore analysis to provide chemical compoisition of the extract and fractions was conducted by with HPLC. The chromatograms of each sample were converted into excel file and then were correlated to its antibacterial activity. There are several possible plots as OPLS outputs to be used to interpret the results. In this study we used score plot, Y-related coefficient plot, and X varian plot. The score plot showed good separation between active and non-active takokak extracts and fractions therefore the study was promising to conduct further. Next, the OPLS Y-related coefficient plot was evaluated. In this study, we are interested to find peak of 17,76 – 18,08 min retention time with a high positive correlation value, which means that the higher peak intensity or the higher peak area value implicates to the higher antibacterial activity as well. In further analysis, we used X varian plot to identify the extract or the fraction where the peak of 17,76 – 18,08 min retention time was dominant. It was discovered that the peak was dominant in fractions K measured at UV λ = 310 nm. Therefore, we chose this fraction for further bioactive identification step with semi-preparative HPLC and LC-MS analysis. The peaks were identified as Torvoside A, Torvoside G, and Torvoside H, three steroidal glycosides. Torvoside A, Torvoside G, and Torvoside H were shown to be abundant in Solanum torvum Swartz fruits active fraction which against Staphylococcus aureus, indicating that Torvoside A, Torvoside G, and Torvoside H might be the responsible compounds for antibacterial activity of the fraction.