Beta-beta wood is the general name for plants of the genus Lunasia, and family Rutaceae. The family Rutaceae contains essential oils, alkaloids, amides, coumarin, flavonoids, benzoic acid, tannins, lignin, phenolics, terpenes, pentacyclic saponins, carbohydrates, scopilatin and mucin [1].

Scopoletin () is known to have anti-inflammatory properties. It is derived from coumarin, a phenylpropane compound [2]. Research on the anti-inflammatory activity of scopoletin has revealed that it inhibits cyclooxygenase and suppresses pro-inflammatory Tumor Necrosis Factor alpha (TNF-α) and Interleukin (IL) - 1β [3,4]. Compounds with basic coumarin skeleton ring structure reduce trauma or disease-induced tissue inflammation due to their antioxidant, anti-inflammatory and immunosuppressive activities [5].

The present study focuses on the anti-inflammatory potential of beta-beta timber extracts, and identification of the bioactive compounds via TLC Scanner and UV-visible spectroscopy.

Materials

Beta-beta wood (Lunasia amara Blanco.) was collected at Siawung, South Sulawesi province of Indonesia in March 2014. Taxonomic identification of the plant was performed by Drs. Joko Santoso. College of Biology Pharmacy Faculty of Pharmacy, Univesitas Gadjah Mada, Indonesia. A voucher specimen (no. BF/204/Ident/Det/V/2014) was deposited at the herbarium of Department of Pharmaceutical Biology Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia for future reference.

Extraction and isolation of compounds

Beta-beta (Lunasia amara Blanco.) wood powder (1 kg) was extracted by maceration at room temperature with 96 % ethanol for 72 h. The maceration process was repeated three times with the same solvent. The extract was collected and evaporated in a vacuum evaporator to obtain a dense residue which was dried and weighed. The dried ethanol extract was dissolved in distilled water and fractionated with dichloromethane using a continuous liquid-liquid extraction (LLCE). Fractionation was done repeatedly until the last fraction was clear. Each fraction obtained was concentrated and weighed, and then subjected to gravitational column chromatography (GCC) using silica gel G 60 (0.04 to 0.83 mm) with dichloromethane: ethyl acetate (97:3, v/v) as eluent.  KOH (10 %) reagent was used to observe the spots, as well as UV absorption at 366 nm and 254 nm. Fractions with similar R_f values were combined. The fractions were identified by UV-visible spectroscopy and assayed using a TLC scanner. The levels of scopoletin in the ethanol extract and fractions were determined from a scopoletin standard curve. The fractions from column chromatography were identified by UV-Visible spectroscopy in the wavelength range of 200 - 400 nm. Identification of fractions was also done by "spiking". Equal volumes and equal concentrations (0.25 mg/mL) of fractions from column chromatography, and scopoletin were spotted on TLC plates, and after development, absorption spectra were obtained. The area under curve (AUC) was measured for scopoletin standard, and for fractions spiked with scopoletin, by densitometry. The AUCs were then compared.

Yield and identity of chemical components

The yield of the crude extract was 1.54 %. shows the TLC chromatogram of the crude extract and its solvent fractions. and show the UV-Visible spectra of the crude extract and scopoletin. From these results, the main chemical component of Lunasia amara   was identified as scopoletin.

Scopoletin compounds absorb maximally at wavelengths of 231 nm, 255 nm, 299 nm, 340 nm and 347 nm [6]. Results from UV-visible spectroscopy for scopoletin indicate maximum absorption at 213, 228, 255 and 344 nm, while beta-beta wood extract and scopoletin standard exhibited characteristic bands at 344 nm and 336 nm, 299 nm, 255 and 259 nm, 228 nm, 229 nm and 213 nm, respectively. This strongly suggests that the beta-beta wood extract and its fractions consist of scopoletin compounds.

Chromatograms of 96 % ethanol extract and dichloromethane fractions produced prominent and intense blue spots after spraying with 10 % KOH. The appearance of dominant blue spots in chromatograms is used to identify scopoletin compounds.  Scopoletin is a coumarin derivative present in plants with anti-inflammatory potential. Values of AUC increased when scopletin was added to the fractions. This suggests that the fractions are similar in identity to standard scopoletin. The fractions and crude extract also had similar spectra and R_f values with scopoletin. Thus the anti-inflammatory property of beta-beta wood (Lunasia amara) is due its content of scopoletin. Numerous studies have demonstrated the anti-inflammatory potential of scopoletin and scopoletin compounds [7,9].

Based on the results of TLC and UV-visible spectroscopy, it seems that the active anti-inflammatory principles in beta-beta wood (Lunasia amara Blanco) are scopoletin compounds.