Advanced Materials Research Vol. 506 (2012) pp 603-606 © (2012) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/AMR.506.603
Characterization of Eugenol Extracted from Lemongrass (Cymbopogon citratus (DC.) Stapf) for Food Packaging Materials N. Petchsoongsakul1,a, C. Pechyen1,2,b 1
Department of Packaging and Materials Technology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand 2 Center for Advanced Studies in Tropical Natural Resources, National Research University-Kasetsart University, Kasetsart University, Bangkok 10900, Thailand (CASTNAR, NRU-KU, Thailand) a
[email protected],
[email protected]
Keywords: Anti-Oxidant, Lemongrass, Eugenol
Abstract. The goal of this research was to study a prospect of applying Lemongrass (Cymbopogon citratus (DC.) Stapf) extracted having antioxidant property (i.e. eugenol) to replace commercial antioxidant agent such as butylated hydroxytoluene (BHT) in packaging raw materials. The extracted eugenol was characterized using Gas chromatography (GC). A free radical scavenging activities of eugenol and BHT were also investigated using 2, 2-dipheny l -1-picryhydrazyl (DPPH) assay. It reveals that a concentration of eugenol resulting in a 50% inhibition of the free radical, IC50, (0.11 mg/ml) is lower than the IC50 value of BHT (0.14 mg/ml) indicating better radical scavenging activity. In its radical form, DPPH• shows an absorbance maximum at 515 nm which disappears upon reduction by an antiradical compound. BHT, a synthetic antioxidant, slowly reacts with DPPH• reaching a steady state within 5 hr. The kinetic (R2) is estimated to be 0.9283 at 25 °C. Eugenol rapid reacts with DPPH• reaching a steady state within 2 h. The kinetic (R2) is estimated to be 0.9946 at 25 °C, our results confirm that eugenol can we used instead of BHT. Introduction Plastic packaging has a widely used to produce film for food packaging. Butylated Hydroxyl Toluene (BHT), is added during plastic processing to protect plastics from oxidative degradation [1]. Because of its high-volatility, it is susceptible to loss through volatilization in high-temperature processing. Their safety, however, has been questioned. At high doses, BHT may cause internal and external hemorrhaging, which leads to death in some strain of small animals. There is much interest among food manufacturers in natural antioxidants, to act as replacements for synthetic antioxidants currently used [2]. It is well known that lemongrass a phenolic compound 4-allyl-2-methoxyphenol, commonly called eugenol with a chemical formula of (C10H12O2). This compound acts as natural antioxidant on oleogenous foods. Eugenol also acts as an anti-carminative, anti-spasmodic and antiseptic in pharmacy and as an antimicrobial agent [3]. Lemongrass (Cymbopogon citratus (DC.) Stapf) are cultivated in many parts of Thailand. With a view to substituting this natural antioxidant compound for synthetic chemicals to avoid the possible toxicity, lemongrass were chosen for eugenol extraction. The objective of this work was to investigate extraction characteristics and to determine optimal conditions for more efficient extraction of eugenol from lemongrass which is primary antioxidant or free-radical chain breaker like BHT antioxidant.
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Experimental Procedure The apparatus shown in Fig. 1(a) was assembled using a 25-mL round bottom flask as the distillation pot. The distillation pot was charged with 1.045 g of ground lemongrasses and 15 mL of distilled water. The lemongrasses were allowed to soak in the water until thoroughly wetted (about 15 min), then the mixture was distilled, the distillate being collected at the rate of about one drop every 2 – 3 seconds. After about 6 mL of distillate were collected, the distillate was extracted with 2.0 mL of CH2Cl2, then again with (2 x 1.0 mL) of DCM. The DCM extracts were combined, dried over Na2SO4, and evaporated to give the product eugenol as a pale yellow oil (Fig. 1(b)).
(a) (b) Fig. 1 Set up extraction apparatus : (a) Extraction tower and (b) Eugenol separation Scanning the wavelengths in the range between 700 and 250 nm for 100 µm of DPPH solved in methanol resulted in maximum absorption in the visible range at 518 nm. The same maximum of absorption was recorded in 100 µm of DPPH dissolved in mixed ethanol–water solution. 100 µm DPPH solution in ethanol–water was prepared in a standard l =1 cm glass spectroscopic cuvette. After reaching the steady state of absorbance at 518 nm, an antioxidant was added to the cuvette and mixed for 10 s. Concentration after the addition of antioxidants was 5 µm. For gallic acid, quercetin and catechin, the final concentration after addition to the spectroscopic cell was 2.5 µm. The changes in absorbance were read after the same period that is necessary for steady state readings in electrochemical experiments. For some antioxidants with rapid kinetics, the readings were done after 3 min, or after 15 min in case of slow kinetics. GC analysis was performed on a Hewlett-Packard HP 5890 fitted with a fused-silica capillary column (SPBTM-1) of 30 m×0.53 mm I.D., 0.53 µm film thickness (Supelco, Bellefonte, USA). A flame ionization detector was used. The sample (0.2 µl) was injected directly onto the column with a Hewlett-Packard 10-µl syringe. Preliminary analysis was performed on reference samples to determine the optimum operating conditions such as sample sizes, temperature parameters, flow-rates and attenuation according to chromatography operating. The optimum operating conditions for the most satisfactory elution profile, composition and spaced peaks was attained with the GC oven temperature programmed at a rate of 5C°/min from 80°C to 230°C. Detector and injector temperatures were set at 230°C and helium gas flow-rate at 3 ml/min. The same data acquisition was used for the lemongrass ethanol-extract sample. Fourier Transform Infrared (FTIR) spectrometry was carried out to observe the structural interactions of BHT and eugenol. The FTIR spectra of liquid samples were recorded from 4000 to 400 cm-1 at resolution of 1 cm-1 using an FTIR spectrometer (PerkinElmer 1760X). Results and Discussion In the DPPH• free radical method, antioxidant efficiency is measured at ambient temperature and thus eliminates the risk of thermal degradation of the molecules tested. However, the reactional mechanism between the antioxidant and DPPH• depends on the structural conformation of the antioxidant. In a recent paper, modifications of the operating conditions were proposed in order to
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adapt the DPPH• method to each kinetic case. Some compounds react very quickly with DPPH•, reducing a number of DPPH• molecules equal to their number of available hydroxyl groups. However, for the majority of the compounds tested, the reactions are slower and the mechanisms seem to be more complex. It would therefore be useful to build plausible kinetic models in order to obtain a better understanding of the mechanisms involving DPPH• and antioxidants. In this paper, we explore the kinetic behavior of two different antioxidants. All the results of this study are summarized in Fig. 2. With R2 values of 0.9283 for BHT and 0.946 for eugenol, our results confirm that eugenol can we used instead of BHT. Adding DPPHH at the end of the reaction regenerates DPPH•. The higher the DPPHH concentration added, the higher the DPPH• concentration at the steady state. Therefore BHT and eugenol reactions are complete.
(a) (b) Fig. 2 Kinetic of Anti-Oxidant agent : (a) BHT and (b) Eugenol. The chromatogram for the standard mixture is shown in Fig. 3. The gas chromatogram of the lemongrass extract in Fig. 3(a), 3(b) and 3(c) shows three major peaks. Identification of eugenol and other major phenolic compounds was made by comparing the retention time and peak appearance in the lemongrass extract in Fig. 3(c) with the standard mixture and commercial chromatogram in Fig. 3(a) and Fig. 3(b). The retention times and the chromatogram peak areas were reproducible for several different aliquots at 0.2 µl injection of the sample. The three major peaks were identified as eugenol, caryophyllene and eugenol acetate. The eugenol peak, the main constituent and high proportion of the extract is quite significant. The three major phenolic compounds in the lemongrass extract are similar to the compounds in the steam volatile extract of air-dried lemongrass but the ethanol extract chromatogram shows less compounds as it displays, in particular, ethanol-soluble compounds of the lemongrass. Isoeugenol also naturally occurs in lemongrass and, on gentle oxidation, yields vanillin but the isomer compound is not detected in this ethanol extract. No identification of the minor peaks has been done as this is outside the scope of this work.
(a)
(b)
(c)
Fig. 3 Chromatogram of Eugenol: (a) Standard, (b) Commercial and (c) Extracted from Lemongrass Steam distillation of lemongrass produced 0.0890 g of an oil which contained in its IR spectrum the functional groups O-H (at 3520 cm-1), sp2 C-H (3080 – 3000 cm-1), aliphatic C-H (2980 – 2940 cm-1), and both alkene C=C (at 1640 cm-1) and aromatic C=C (at 1514 cm-1) and CO-CH3 (at 3520 cm-1) and C-H (at 818 and 851 cm-1). These data are consistent with the structure
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of eugenol, shown in Fig. 4. Therefore, it can be concluded that the oil which was isolated from lemongrass is in fact, eugenol. 0.0890 g of eugenol was recovered from 1.045 g of lemongrass. This corresponds to a percent recovery of 8.51%: Amt. Eugenol isolated % Recovery = ----------------------------------- = Amt. Lemongrass used
0.0890 g ----------------- x 100 = 8.51% 1.045 g
Fig. 4 FTIR spectrum of eugenol extracted from Lemongrass (Cymbopogon citratus (DC.) Stapf). Although the % recovery seems slightly low relative to the expected 10%, the experiment proceeded as planned. There were no spills or other abnormal physical losses. It is possible that the ratio of the size of the glassware to the theoretical amount of eugenol which can be obtained from lemongrass in this experiment is large, leading to adherence of a large percentage of the product on the sides of the glass apparatus. If this is so, then steam distillation of a larger sample of lemongrass should give an improved recovery. Otherwise, it can be concluded that the specific sample of lemongrass used contains approximately 8.51% eugenol. There has been a marked increase in the literature on antioxidant properties of herbs and spices. Similarly S.Dragland et al. presented contents of antioxidants determined by % recover of eugenol, for example in clove 10.35, oregano 9.60, allspice 8.50, ginger 6.55, black pepper 7.73 and cumin 6.80 in dependence on the extraction techniques used by the authors. The antioxidant activity correlated significantly and positively with total phenolics, DPPH• concentration and percent recovery of eugenol [4]. Conclusions Use of DPPH• provides an easy and rapid way to evaluate the antiradicalar activities of antioxidants, but also to build plausible kinetic models of reactions. From the results obtained in the present study, the reaction mechanisms for BHT and eugenol all showed a hydrogen atom transfer reversible step which reduces the DPPH• and forms the antioxidant radical (A•). The antioxidant activity of phenolic compounds is mainly due to their redox propertie, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quenchers. Acknowledgements This study is financially supported by The Graduate School Kasetsart University and TRF-Master Research Grants from Thailand (MRG545S075). References [1] P. Suppakul, J. Miltz, K. Sonneveld And S.W. Bibber: Journal of Food Science. Vol.68 (2) (2003), p.408-420 [2] W. Nantitanon, S. Yotsawimonwat and S. Okonogi: LWT - Food Science and Technology. Vol.43 (2010), p.1095-1103 [3] U. Siripatrawan and B.R. Harte: Food Hydrocolloids. Vol.24 (2010), p.770-775 [4] S. Dragland, K. Holte, R. Blomhoff: Journal of Nutrition, Vol.133 (2003), p. 1286-1290
