CHARACTERIZATION OF ETHANOLIC EXTRACT FROM PROPOLIS PRODUCED BY PHILIPPINE STINGLESS BEES Trigona biroi Friese HERVIN ERROL T. MENDOZA1, Hidelisa P. Hernandez2, Archie C. Reyes, Banyuhay P. Serrano 1Institute of Chemistry, College of Arts and Sciences, University of the Philippines Los Baños, College, Laguna; hervinerrol@yahoo.com 2Institute of Chemistry, College of Arts and Sciences, University of the Philippines Los Baños, College, Laguna; heidi_phern@yahoo.com Introduction Results and Discussion Propolis is made up of resinous material and usually varies in color such as cream, yellow, red, green, light and dark brown. The word propolis comes from the Greek word pro, which means “in the defense of” and polis which literally signifies “city”. From its meaning, the function of propolis is to protect the bee community. Researches in the chemistry of propolis has been the interest due to its potential bioactivity (Bankova, 2005). In the Philippines, honeybees are the main sources of propolis which are being exported to other countries. Continuous expansion of this market may no longer be met by honeybee propolis, so stingless bee (Trigona sp.) propolis started to be mass produced. One of the attempts done in propolis research was isolation of constituents by solvent extraction and identification of the compounds present. Analysis of stingless bee propolis in the Philippines must be employed before its recommendation for medical uses. Chemical constituents must be identified and studied for potential therapeutic applications, particularly to human health and for the development of its commercial uses. Propolis started to soften at 40-45 oC and melted at around 85 oC. Residue from extraction also softens at 40-45 oC and melted at 60-65 oC. This showed that low-melting compounds in propolis still remain after extraction with 70% ethanol. Results also showed that residue from extraction melted at relatively lower temperature than propolis. Folin-Ciocalteau analysis showed that AQ has the greatest amount of phenolic content. a Source: Shriner, R. L. et al. 2004. The systematic identification of organic compounds. 4th ed. USA: John Wiley and Sons, Inc. p. 165. NMR analysis of DF suggests that probable compounds present are hydrocarbons and oxygenated organic compounds such as alcohols and ether. Assuming all the components have similar response factors, the most abundant components of HF are 1-heptatriacontanol (C37H74OH), tetratetracontane (C44H90) and nonadecane (C19H40). The major components of HF mainly consist of straight-chain hydrocarbons and long-chain alacohols. Table 1. Amount of ethanol extract (EE) and the hexane soluble fractions (HF), dichloromethane soluble fractions (DF) and aqueous fractions (AQ) based on EE. EE HF DF AQ 10.01 ± 0.16% 2.99 ± 0.19% 39.39 ± 3.04% 57.62 ± 0.03% Table 2. Estimated amount of phenolic contents in hexane soluble fractions (HF), dichloromethane soluble fractions (DF) and aqueous fractions (AQ) by Folin-Ciocalteau method expressed as gallic acid equivalent (GAE). Fraction HF DF AQ mg GAE/ 100 g 109 ± 2 744 ± 11 2413 ± 34 Objectives Table 3. Probable chemical compositions of dichloromethane soluble fractions (DF) with their corresponding chemical shifts for protons analyzed by proton NMR. The specific objectives of this study are to extract the ethanol soluble components of stingless bee propolis; determine the melting temperature of propolis and residues after extraction; estimate the phenolic content of hexane soluble fractions , dichloromethane soluble fractions and aqueous fractions of ethanol extract by Folin-Ciocalteau colorimetric method; predict the chemical composition of dichloromethane soluble fractions by nuclear magnetic resonance analysis; and identify components of propolis present in the hexane and dichloromethane soluble fractions using gas chromatography–mass spectrometry. Chemical Shift of H (ppm) Possible Classification of Compounda 0.74 – 1.86 Alkane 3.70 – 3.74 Alcohols, Ethers 4.00 – 5.30 Alkene Table 4. Tentative identity of compounds identified from hexane soluble fractions (HF) with the corresponding retention times analyzed by GC-MS. Methodology Retention Time (min) Percent Peak Area (%) Tentative Identity 17.08 1.363 9-Octadecenal 18.02 0.775 2-butyl-1-octanol 18.95 3.476 Octacosane 19.84 7.230 Nonadecane 20.75 13.356 Tetratetracontane 21.80 9.246 Heneicosane 23.05 7.122 6-methyloctadecane 24.55 3.013 Hexadecane 26.43 1.652 2,4,6,8-tetramethyl-1-undecene 28.88 46.336 1-heptatriacontanol 29.54 6.429 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol Extraction with 70% Ethanol for 24 hours 1 Fractionation of Ethanol Extract with Hexanes and Dichloromethane Gas Chromatography-Mass Spectrometry (GC-MS) of Hexane Fraction 2 6 Nuclear Magnetic Resonance (NMR) of Dichloromethane Fraction 3 5 Heating of Propolis and Residue 4 Estimation of Phenolic Content by Folin-Ciocalteau Method Summary and Conclusion Literature Cited Preliminary study on the characterization of Philippine stingless bee Trigona biroi Friese propolis by extraction with 70% ethanol aided with spectroscopic analyses showed that ethanol extract consists of phenolic compounds, hydrocarbons and long-chain alcohols. Aqueous fraction of the ethanol extract was found to have the highest phenolic content. Further study must be employed to be able to understand and generalize the characteristics and properties of compounds present in the ethanol extract of propolis. BANKOVA, V. S. 2005. Recent trends and important developments in propolis research. eCAM 2005; 2 (1): 29-32. SHRINER, R. L., HERMANN, C. K. F., MORILL, T. C., CURTIN D. Y. FUSON, R. C. 2004. The systematic identification of organic compounds. 4th ed. USA: John Wiley and Sons, Inc. pp. 260-261.