Physicochemical and Antioxidant Properties of Cymbopogon citratus Essential Oil

In this study, the essential oil (EO) of lemongrass (Cymbopogon citratus) or “lemon tea” leaves was studied. The EO was obtained by the steaming (0.75 ± 0.05%) and distillation (1.5 ± 0.07%), assisted by microwaves, methods. The EO had a refractive index of 1.483 ± 0.001 (20 °C) and a density of 0.873 ± 0.005 g/mL (27 °C). Color parameters of the oil corroborate the yellow hue observed by the naked eye. The Gas chromatography-Mass spectrometry (GC-MS), Fourier Transform Infrared (FT-IR) Spectrometry, and Nuclear Magnetic Resonance (NMR) techniques permitted to characterize the EO and revealed the chemical structure of the major component: citral (neral and geranial). The phenolic compounds content was 149.2 ± 6.0 mg Gallic acid equivalents (GAE) per 100 mL of oil and the antioxidant activity was 44.06 ± 0.20 mg Trolox (T) per 100 mL of essential oil.

The scientific name of lemongrass is Cymbopogon citratus.The Cymbopogon word derives from the Greek words "kymbe" (boat) and "pogon" (beard), referring to the arrangement of the spike of the flower.The word citratus derives from the old Latin, meaning lemon-scented leaves (Shah et al., 2011).The common name of Cymbopogon citratus in Mexico is "zacate limón" (lemongrass) or "té limón".It is a perennial tropical grass; is resistant to different temperatures and can grow in warm, semi-warm and temperate climates.It is from 60 to 120 centimeters high, its leaves are green, long and slats and have pleasant aroma and taste.This grass is native to India (Parikh & Desai, 2011).Because of its pleasant flavor, in Mexico is consumed as infusion in water or milk just because the herbs intake is a custom in the Mexican population (Juárez-Rosete et al., 2013).The essential oil of Cymbopogon citratus has shown to have anti-inflammatory, analgesic and antipyretic properties, (Gbenou et al., 2013) besides having antimicrobial effects (Hammer, Carson, & Riley, 1999;Pranoto, Salokhe, & Rakshit, 2005;Adukwu, Allen, & Phillips, 2012).Tzortzakis & Economakis (2007) reported that the essential oil of lemongrass inhibited the growth of Botrytis cinerea.Later Raybaudi-Massilia, Rojas-Graü, Mosqueda-Melgar, and Martín-Belloso (2008) reported that the oil of lemongrass could suppress the growth of mesophiles and psychrophiles in fresh-cut apples.
The aim of this study was to obtain and characterize the essential oil of Cymbopogon citratus.

Plant Material
Fresh Cymbopogon citratus leaves were acquired in the Puebla City Central Market, Puebla, Mexico.

Essential Oil Extraction
Fresh Cymbopogon citratus leaves were dried at room temperature for one week; leaves were extended on trays, turning them three times daily for ventilation, accelerate drying, and preventing the growth of microorganisms.The essential oil of Cymbopogon citratus was obtained by two methods.Hydrodistillation: 50 g of ground plant material was extracted for about 60 minutes (Baizabal, 2010) using a Clevenger apparatus.Microwave assisted extraction: 50 g of ground leaves and 200 mL of distilled water were placed in a 500 mL spherical flask; the distillation was performed for about 30 min.The distillation apparatus was placed inside of a conventional Daewoo DC (model KOR 6LYB) microwave oven.A power of 600 W was used.The yield of oil was calculated by the following equation: Yield (%) = (Oil (mL))/(Plant (g)) × 100

Refraction Index
To measure the refractive index of oil, an Atago ND R5000 refractometer (Osaka, Japan) was used according to the 921.08 AOAC (2000) method.

Density
It was assessed according to the 985.19 AOAC (2000) method using a 2 mL pycnometer.

Color
The CIELAB color parameters were measured using a Colorgard 05 colorimeter (Gardner Laboratory, USA) in the transmittance mode.The equipment calibration was performed with the black mosaic and the calibration parameters: L* = 100, a* = 0, and b* = 0.A cell of quartz and a volume of 3 mL of oil was used.

Gas Chromatography-Mass Spectroscopy
It was performed using an Agilent Technologies 6850 gas chromatograph (Santa Clara, CA, USA) coupled to an Agilent Technologies 5975 mass selective detector (Santa Clara, CA, USA).An Agilent capillary column (HP-5 ms nonpolar 5 % phenyl methyl polysiloxane) of 30 m, 0.25 mm in diameter and 0.25 microns in thickness was used.Helium, at a flow rate of 1.1 mL/min, was used as the carrier gas.The oven temperature of the gas chromatograph was maintained at 300 °C.The temperature in the column was started at 60 °C and maintained for 2 min, then increased, until reaching 250 °C, at a rate of 10 °C/min.Therefore, the final temperature was maintained for 10 min.One microliter of oil was injected into the column using a split injection of 10:1.The injector temperature was 250 °C (Conde-Hernández & Guerrero-Beltrán, 2014).The components were identified by their fragmentation patterns of mass spectra compared with data stored in the National Institute of Standards and Technology Mass Spectral database (NIST-MS, 2010).

Fourier Transform Infrared (FT-IR) Spectrometry
Spectrometry of the EO of Cymbopogon citratus was carried out with a Spectrum One FT-IR Spectrometer, (PerkinElmer ® , Waltham, Massachusetts, USA).An EO sample was placed directly on the surface of the ATR top plate at room temperature; measurements were performed in the IR region at 4000-650 cm -1 .Four scans were performed at a resolution of 4.00 cm -1 at a scan speed of 0.20 cm/s.The crystal used was diamond SSNS.An air spectrum was used as reference (Wany et al., 2014).

Phenolic Compounds
The Folin & Ciocalteu method (Singleton, & Rossi, 1965) was used for determining phenolic compounds with some modifications.Fifty microliters of EO were diluted with 950 µL of ethanol.From this dilution, 500 µL were taken and added with 3 mL of distilled water and 250 µL of Folin-Ciocalteu reagent.The reaction was stopped adding 750 µL of 20 % Na 2 CO 3 .Finally, the solution was brought to 5 mL with distilled water.The reaction was left in the dark for 2 hours.Samples were filtered through Whatman paper No. 4 before recording absorbances.A blank was prepared with 4 mL of distilled water, 250 µL of Folin-Ciocalteu reagent, and 750 µL of 20% Na 2 CO 3 .The absorbance was recorded at 765 nm using a UNICO ® 2800H UV-visible spectrophotometer (Shanghai, China).Phenolic compounds were expressed in mg equivalent of Gallic acid per 100 mL of oil.The measurements were performed in triplicate.The phenolics concentration was calculated from a standard curve of Gallic acid (0-0.0672mg): Abs = (15.03/mg)*[mg]+0.0886(R 2 = 0.973).From a standard solution of Gallic acid (0.186 mg/mL), nine tubes were prepared to obtain concentrations of 0 to 400 µL.

Antioxidant Capacity (AC)
The method used to determine antioxidant capacity of the essential oil was adapted from Brand-Williams, Cuvelier and Berset (1995) with some modifications.Radical solution: it was prepared dissolving 3.94 mg of DPPH (2,2-Diphenyl-1-picrylhydrazyl) (Sigma-Aldrich, Toluca, Edomex, Mexico) in 100 mL of ethanol.AC: Fifty L of EO was mixed with 950 L of ethanol, 2 mL of DPPH radical solution, made up to 4 mL with ethanol, and thoroughly mixed.The mixture was left to react in the dark for 30 minutes.The absorbance of samples (A s ) was recorded at 517 nm in a UNICO® 2800H UV-visible spectrophotometer (Shanghai, China).A blank (A b ) was prepared using the same procedure substituting only the sample for ethanol.The percentage of inhibition (I) was calculated as follow: The antioxidant capacity (mg T/100 mL of EO) was calculated from a standard curve of Trolox (0-0.028mg): I = (2699.2/mg)*[mg]-4.3749(R 2 = 0.989).To prepare the calibration curve a standard solution of Trolox ((±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, 97 %) was prepared (0.35 mg/mL) in ethanol.Eight tubes were used to obtain concentrations in the range 0 to 0.028 mg of Trolox.

Statistic Analysis
An analysis of variance and Tukey test were performed to state differences between means using the Minitab 17 software (LEAD Technologies Inc., NJ).A P value of 0.05 was considered statistically significant.

Essential Oil Yield
Yields of 0.752 ± 0.05 and 1.5 ± 0.07% (V/W) of EO were obtained by the steam and microwave assisted distillation methods, respectively.Differences (p < 0.05) within means of yields were observed.Very probably the core of ground plant placed in the spherical flask was efficiently heated due to microwaves.As observed in other works, when using a simple distillation procedure, sometimes the steam do not reach the center of the ground plant; therefore, the yield of the oil extraction is not optimized.The moisture content of the dried C. citratus was 9.14 ± 0.70%.

Physical Properties
The values of refractive index, color, and density are shown in Table 1.These values are close to those reported by Tovar et al. (2011) ( = 0.885 g/mL; IR = 1.482),Paviani, Pergher, and Dariva (2006)   The predominant component of the essential oil of C. citratus obtained by the two distillation methods of extraction was citral (mixture of geranial and neral aldehydes) (Figure 1).These results agree with those reported by Mahanta et al. (2007) and Negrelle and Gomes (2007).Adukwu, Allen, and Phillips (2012) Ruangviriyachai, and Luthria (2012) reported that the plant has a content of citral of 65 to 80%.Mohamed Hanaa, Sallam, El-Leithy, and Aly (2012) reported concentrations of geranial (31.53%, 39.86% and 37.24%), neral (30.08%, 34.52% and 31.28%) and myrcene (16.61%, 14.49% and 15.42%) in essential oils extracted from leaves of Cymbopogon citratus, dried in the sun, in the shade, and in oven, respectively.The differences in type of components reported by other researchers is probably due to the climatic conditions where the plant was grown.The quality of Cymbopogon citratus is determined by the content of citral.It has been observed that citral has an inhibitory effect on bacteria and fungi (Adinarayana et al., 2012;Marques et al., 2013).
. Chemical structures of geranial and neral

Infrared Spectrum
As mentioned above, the main component of the C. citratus EO is citral.In the IR spectrum (Figure 2) of EO of C. citratus, the functional groups of citral were observed.In the vibrations at 2968 cm -1 , a predominant asymmetric stretching of -CH 3 is observed corresponding to an alkyl saturated aliphatic group and at 2915 and 2857 cm -1 , symmetric and asymmetric stretching of -CH 2 are observed.The intense band observed at 1671 cm -1 is due to vibrations of C=C (cis and trans), confirming the presence of conjugated double bonds (C = C-CHO) in citral which are common in acyclic monoterpenes.The peak at 1632 cm -1 indicates stretching of C=O of the aldehyde group.At the 1442 cm -1 peak, bending of the -CH 2 group is observed.At 1377 cm -1 bending of -CH 3 group is observed.From 1194 to 1120 cm -1 , stretching of -C-O and vibrations of the -CH skeleton are observed.At 841 cm -1 , substitution in 1,3 and 1,4 are observed.Similar peaks were reported by Wany et al. (2014) (= 0.848 g/mL),Essien, Essien, Ita, & Ebong (2008) ( = 0.888 g/mL; IR = 1.477),Ibrahim, Ibo and Adejare (2010) ( = 0.866 g/mL; IR = 1.472), andMonteiro et al. (2011) ( = 0.949 g/mL; IR = 1.332) who reported similar refractive index and specific gravity values for essential oils of lemongrass, lemongrass, citron (Citrus medica L.), ginger, and allspice (Pimenta dioica), respectively, obtained by hydrodistillation.Color parameters indicate a yellow hue (68.81 ± 0.36) which was observed in the EO.Tovar et al. (2011) reported high values for L* (98.59 to 101.63) but similar values for a* (-2.45), and b* (6.28) color parameters for the Cymbopogon citratus EO.

Table 1 .
Refractive index, density, and color parameters of dried C. citratus EO obtained by distillation assisted by microwaves

Table 3 .
Chemical compounds of essential oil of dried C. citratus obtained by distillation assisted by microwaves and Andrade et al. (2012)informed that citral was the main component in C. flexuosusu and C. nardus, respectively.The highest percentage (51.63%) of citral in the EO obtained by the microwave assisted distillation method is close to that reported byLeón-Anzueto et al. (2011) (59.7to 64.0%) in C. citratus (DC.)Stapf.Chanthai, Prachakoll, for geranial from other species of Cymbopogon winterianus (citronella grass).