Isolation, Identification And Antioxidative Activity Of Phenolics In Palm Oil Mill Effluent

Author

NORMALA BT HALIMOON

Date

2006

Keyword

palm oil mill effluent, phenolic compounds, antioxidative activity, free radical scavenger activity

Abstract

The objectives of the study are to isolate, and identify the phenolic compounds present in the palm oil mill effluent (POME) and evaluate their antioxidative activity. Combinations of column and partition chromatography were used in the isolation of the phenolic compounds. Antioxidative activities of the POME fractions were evaluated for free radical scavenging activity, lipid thiocyanate, ß-carotene-linoleate and reducing assays. Identification of the phenolic compounds in POME filtrate was carried out using high performance   liquid   chromatography   (HPLC),   followed   by   confirmation   of the   compounds   using   gas   chromatography mass   spectrometry   (GC/MS)   and   liquid chromatography mass spectrometry (LC/MS). The study revealed the presence of cinnamic acid derivatives, which were measured at 320 run. These included chlorogenic, p-coumaric, caffeic, ferulic acids and rutin hydrate. Benzoic acid derivatives, which were identified as protocatechuic, gentisic, p-hydroxybenzoic and vanillic acids were also present when measured at 260 nm. Hydrolysis of the sample did not affect the chromatographic pattern obtained indicating the stability of the phenolic compounds in the POME filtrate. Flavan-3-ol groups (catechins) were identified from ytterbium precipitation method. Four fractions have been isolated from POME filtrate based on flash chromatography coupled with uv/vis detection. The antioxidative activity of each fraction was then evaluated. All the POME fractions demonstrated excellent antioxidative activity in all the assays used. This is especially true for both Fractions 3 and 4. Fraction 1 exhibited highest antioxidative activity based on reducing assay followed by Fraction 3 indicating the excellent metal chelating ability of the phenolics in these fractions. The POME fractions were found to contain different levels of total phenolics, which did not necessarily correspond to the strength of antioxidative activity, measured for each fraction. Fraction 3 showed strong free radical scavenger and phenolic content was also high. Fraction 2 showed low antioxidant activity, while total phenolic content was low. However, Fraction 4 showed high antioxidant activity with low total phenolic content This indicates the presence of different phenolic compounds in the different fractions. Results showed that Fraction 1 contained an unknown polar compound and protocatechuic acid with strong metal chelating activity, Fraction 2 was rich in p-Tiydroxybenzoic acid, Fraction 3 contained three unknown cinnamic acid derivatives and {Fraction 4 contained known cinnamic acid derivatives compounds including p-coumaric acid, rutin hydrate and ferulic acid with lower polarity. The cinnamics in Fraction 3 consistently exhibited higher antioxidative properties compared with benzoics in Fraction 2. Combination study of the fractions revealed that mixture of Fraction 3 and ascorbic acid had better free radical scavenging activity. However, the synergistic effect was observed only at low concentration of 2.5 and 5 mg/1 of Fraction 3 and ascorbic acid. The individual compounds of Fraction 3 (cinnamic acid derivatives) including Compounds 14, 15 and 16 exhibited the highest free radical scavenging activity. Highest antioxidative activity was also measured when Compounds 15 and 16 were combined (double combined) while for triple combinations, the highest activity was seen with Compounds 3, 14, and 15 at 15 mg/1 of total phenolic contents. The results showed that there was a synergistic effect between the compounds in particular those in Fraction 3. GC/MS and LC/MS were employed to confirm identify of unknown compounds in Fraction 3. Three compounds in Fraction 3 were observed to exhibit similar absorbance spectra using HPLC with diode array detection. LC/MS revealed presence of dimer with maximum molecular weight of 335 m/z. GC/MS revealed the presence of compounds with a general cinnamic acid structure and derivatives with silylate group. Antioxidative activity of the compounds was in the following order; Compound 14 > Compound 16 > Compound 15. Compounds 14,15 and 16 were tentatively identified as 2,4-dihydroxycinnamic acid, 2,5-dihydroxycinnamic acid and 2,3-dihydroxycinnamic acid.