Identification of active anti-inflammatory principles of beta-beta wood ( Lunasia amara Blanco ) from Siawung Barru-South Sulawesi , Indonesia

Purpose: To identify the anti-inflammatory components of beta-beta (Lunasia amara Blanco.) wood. Methods: The wood material was extracted with 96 % ethanol and fractionated with dichloromethane using a liquid-liquid continuous extraction (LLCE). The fractions were subjected to silica gel column chromatography. Components of the extracts were identified by thin layer chromatography (TLC) scanner and UV-visible spectroscopy, using scopoletin as standard. Results: TLC results for Lunasia amara extract showed the same spot as standard scopoletin. UVvisible spectrum for scopoletin displayed maximum absorption at 213, 228, 255 and 344 nm, while betabeta wood extract showed characteristic bands at 344, 336, 299 and 255 nm. The results indicate that the main components of the extracts are scopoletin and its derivatives. Conclusion: The active anti-inflammatory compound in beta-beta (Lunasia amara) wood is scopoletin.

Research on the anti-inflammatory activity of scopoletin has revealed that it inhibits cyclooxygenase and suppresses proinflammatory Tumor Necrosis Factor alpha (TNFα) 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 antiinflammatory potential of beta-beta timber extracts, and identification of the bioactive compounds via TLC Scanner and UV-visible spectroscopy.

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 %. Figure 2 shows the TLC chromatogram of the crude extract and its solvent fractions.Figure 3 and Table 1 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.

DISCUSSION
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].

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

Figure 1 :
Figure 1: Chemical structure of scopoletin (7hydroxyl -6 -methoxy coumarin) EXPERIMENTAL 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.

Figure 3 :
Figure 3: UV-Vis spectra of Beta-beta wood extract fractions

Table 1 :
UV-Vis spectra of beta-beta wood extract fractions and standard scopoletin