Antibacterial Activity of Scopoletin from Stem Bark of Aleurites moluccana Against Salmonella typhi

In antibacterial screening by diffusion test, methanol extract of stem bark A.moluccana showed excellent growth inhibition of Salmonella Typhimurium cause of typhoid fever. Some coumarin metabolites are very good as an anti-bacterial against Salmonella Typhimurium. Method: Simplicia of A.moluccana stem bark was extracted by maceration using methanol. The dry extract is extracted liquid using water-organic solvent (hexane and ethyl acetate) in a separating funnel. The hexane and ethyl acetate fraction was monitored for active spots with TLC bioautography. The active compound is separated using vacuum liquid chromatography and radial chromatography. Results: The fraction J of KCV and J2 subfraction results from radial chromatography showing the antibacterial activiiuty of S. thpimurium. From the J2 subfraction obtained pure isolates in the form of yellowish needle crystals. The isolate was tested for antibacterial S.Typhimurium using the microdilution method with a value of MIC is 250 μg/ml. Based on spectroscopic data and comparing the published spectra of the compound, the elucidation of the isolate is Scopoletin (7-hydroxy-6-methoxycoumarin).

paratyphoid fever, and a large number of serotypes are responsible for the severe gastrointestinal disease. The serotypes can be distinguished by agglutination and staining. Aleurites moluccana bark is used by the people of East Kalimantan Indonesia for antibacterial drugs for typhoid fever. [3][4] .
Previous studies have shown the antibacterial activity against some Isolates clinical isolates from A. moluccana extracts which may be caused by the composition of polyphenols, including as a coumarin group [5]. Some coumarin metabolites such as scopolatin are very good as antibacterial. [6][7][8]. This study wanted to show the antibacteria activity of the content of the scopolatin from A.molucana against Salmonella Typhimurium that causes typhoid fever.

Samples
The plants used in the study were A.moluccana stem bark. Sample used in the study was obtained from Topeng village, Klaten district, Central Java. Determination of plants is done at the Herbarium of the The School of Life Sciences and Technology, Bandung Institute of Technology. Sample preparation includes, selection of healthy skin (wet sorting), washing, drying, grinding, sifting to obtain dry powder ready for extraction.

Antibacterial with Bioautography Test
In the test sample antibacterial activity using TLC was carried out using the silica gel GF254 plate (Meck®). MHA media are mixed with bacterial test suspension. The chromatographic results were placed on the medium for 30 minutes then released. Incubated (Wise Cube® WIG-105) at 37 °C for 24 hours. Antibacterial activity is indicated if there is a clear zone in the media. The distance of RF is confirmed by the results of Thin Layer Chromatography (Camag® ) to localize stains that have activity [11].

Microdilution Test
Microdilution test was carried out with test hole microplate (iwaki®). The concentration used starts from 1000, 500, 250, 125, 62.50 and 31.25 µg/ml. The test sample was dissolved with NaCl sterile with the help of DMSO. All holes in the microplate are filled with 100 µL MHB media. The test hole was filled with 100 µL bacterial suspension. The negative control (DMSO) and bacterial control was also carried out at different holes. Incubated at 37 °C for 24 hours. The experiment was repeated three times. Minimum Inhibition Concentration is determined by looking at turbidity and sediment [11][12].

■ Results and Discussion
The skin of A.moluccana stem bark obtained carried out by drying and grinding to obtain simplicia powder. Some stages of chemical separation of A.moluccana bark include maceration using methanol, fractionation using hexan and ethyl acetate, vacuum chromatography, and radial chromatography [13]. The initial profile of activity against S.typhimurium bacteria was used TLC bioautography. Antibacterial activity in the J fraction and sub J2 fraction has antibacterial activity. The compound on the TLC plate diffuses into the media so that direct contact with bacteria occurs. The active J fraction inhibits the S.Typhimurium bacteria in the form of a clear area which has the same RF as the chromatogram profile on blue fluorescent spots under UV 254 or 366 nm shown in Figure 1.  In the J2 fraction, recrystallization was carried out, obtained by the pure compound in the form of yellowish-white needle crystals. Previous isolates were obtained and characterized using chemical tests, UV-Vis spectrophotometers and nuclear magnetic resonance (1D-NMR and 2D-NMR), as well as literature. The isolate is a scopoletin (7-hydroxy-6-methoxycoumarin), which has been identified in previous studies. [13].
Quantitative testing of minimum inhibitory concentration (MIC) of isolates by microdilution method using a well microplate test. The profile of antibacterial activity in MHA media is seen as a clear zone. Scopoletin MIC against S.Typhimurium bacteria (Figure 3) was found at a concentration of 250 µg/ml which occurred in all three replications. Antibacterial activity is indicated by the absence of sediment and turbidity. MIC values have been confirmed by scraping samples of 250 µg/ml and 125 µg/ml on MHA media. Bacterial growth is only found at a concentration of 125 µg/ml.
Coumarin is composed of heterocyclic form of benzene and pyrone rings substituted for oxygen and their derivatives with strong biological activity. Coumarin is considered a heterocyclic bioactive mixture which promises various anti-microbial activities because it can damage the cell wall permeability [7,14,15].
Scopoletin compound has been known to have activity against the bacteria Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus and Staphylococcus aureus with MICs +/-1000 µg/ml [16]. In testing the bacteria S.Typhimurium (U937) which is resistant to human intracellular macrophages invitro also shows good results