INTRODUCTION

Cymbopogon citratus (D.C.) Stapf., commonly known as lemongrass, belongs to family Poaceae and is widely cultivated in many tropical and subtropical regions due to its aromaticand medicinal properties. In Ecuador, this grass with lemon scented property is commonly named “hierba luisa”. The essential oil (EO) obtained from the leaves is widely used in perfumery, food and pharmaceutical industries due to its high citral (a mixture of isomers neral and geranial) content which are responsible of the strong lemony aroma and the antimicrobial activity, including the inhibition of pathogenic and spoilage microorganisms (Menut et al., 2000; Appendini & Hotchkiss, 2002; Raybaudi-Massilia, Mosqueda-Melgar & Martín-Belloso, 2006; Tzortzakis & Economakis, 2007; Raybaudi- Massilia et al., 2009; Naik et al., 2010; Matasyoh et al., 2011; Leite et al., 2016; Athayde et al., 2016; Ekpenyong & Akpan, 2017).

In Ecuador, C. citratus is commercially grown in the Amazonian region, where its leaves are used as a food seasoning and for preparation of tea. However, only one study has been done about the chemical composition of its EO and biological activities (Sacchetti et al., 2005). Therefore, the present study was done to analyze the chemical composition and biological activities of the EO from lemongrass (Cympopogon citratus[D.C.] Stapf.) leaves grown in Amazonian Ecuador.

MATERIALS AND METHODS

Materials and isolation of essential oil

Leaves of lemongrass were collected by Fundacion Chankuap' (Macas, Ecuador) in May 2017 from wild trees on the outskirts of the Wasak'entsa reserve in eastern Ecuador and positively identified by the National Herbary of Pontificia Universidad Católica del Ecuador (voucher nr. HERUTEQ1056). Fresh leaves (300 g) were steam distilled for 6 h in a pilot- scale distiller. EO yield was 0.3 % v/m.

Gas chromatography

Analyses of the EO was performed by gas chromatography with a flame ionization detector (GC-FID) on a Konik 4000A (Konik, Barcelona) equipped with a 30 m x 0.25 mm i.d x 0.25 mm DB-5ms (J & W Scientific, Folsom, CA, USA) column. The GC parameters were: oven temperature program, 70 oC (2 min), 70 - 240 oC (4 oC/min) and 240 oC (5 min); carrier gas hydrogen flow rate 1 mL/min; injector and detector temperatures 250 oC, Samples (1 μL) were injected using split ratio 1:100, and previously diluted in n-pentane (1:6 v/v). The constituents were quantified after the areas of each detected compound were normalized and expressed as a percentage area.

The EO oil was also examined by gas chromatography-mass spectrometry (GC-MS) using a QP-2010 Ultra (Shimadzu, Japan) with a 30 m x 0.25 mm i.d. x 0.25 mm BP-5 (SGE Analytical Science Pty. Ltd., Victoria, Australia) column. Temperature program and Helium carrier gas flow rate same as in GC-FID. EIMS: electron energy, 70 eV; ion source and connecting parts temperature, 250 oC. Acquisition was performed in scanning mode (mass range m/z 35-400 u). Compounds were identified using their linear retention indices and mass spectra. Linear retention indices, calculated using linear interpolation relative to retention times of C8-C24 of n-alkanes, were compared with those standards and data from the literature (Adams, 2001)). Mass spectra were compared with corresponding reference standard data reported in the literature (Re et al., 1999) and mass spectra from NIST 05, Wiley 6, NBS 75 k, and in-house Flavorlib libraries. In many cases, the essential oil was subject to co-chromatography with authentic compounds.

ABTS radical cation decolorization assay

The spectrophotometric analysis of ABTS scavenging activity was determined according to anestablished method (Re et al., 1999). Briefly, the pre-formed radical cation of ABTS was generated by reacting ABTS solution (7 mmol/L) with 2.45 mmol/L potassium persulfate (K2S2O8). The mixture was allowed to stand for 15 h in the dark at room temperature. The solution was diluted with methanol to obtain the absorbance of 0.7 ± 0.1 units at 750 nm. An aliquot of 100 µL of methanolic dilution of EO was added to 1 mL of ABTS free radical cation solution. The absorbance, after 10 min, was measured spectrophotometrically at 750 nm. Methanolic solutions of known concentration of Trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid, a water-soluble vitamin E analog) in the range of 50 to 700 µmol/L were used as calibration curve. The results were expressed as millimol Trolox per 100 mL EO.

Ferric-reducing antioxidant power (FRAP) assay

The FRAP of the EO was measured by the method earlier reported (Benzie, 1996). Briefly, acetate buffer (300 mmol/L, pH=3.6), TPTZ (2,4,6-tripyridyl-s-triazine; Sigma) 10 mmol/L in 40 mmol/L HCl and FeCl3·6H2O (20 mmol/L) were mixed in the ratio of 10:1:1 to obtain the working FRAP reagent. The EO (90 µL) was mixed with 900 µL of FRAP reagent. A solution of ascorbic acid was used as standard and Trolox, a stable antioxidant was used as positive control. The mixtures were incubated at room temperature for four minutes and the absorbance was measured at 593 nm. The FRAP was expressed in units of ascorbic acid equivalent.

Minimal inhibitory concentration

For determination of minimal inhibitory concentration (MIC), 5; 0.5; 0.05 and 0.005 μL/mL of the EO were prepared with dimethyl sulfoxide. One milliliter of each EO dilution was placed in different test tubes followed by addition of 1 mL of 24h–broth culture of the microorganism. The test tubes were all sealed with sterile corks and subsequently incubated at 32 oC for bacteria and 25 oC for fungus during 48 h. After incubation the tubes were observed for clearance or turbidity. The tube with highest degree of clearance was taken as the MIC. Three independent tests were performed for each sample. This procedure was separately carried out for the six test microorganisms: Bacillus subtilis ATCC 6633 (G+), Staphylococcus aureus ATCC 25923 (G+), Escherichia coli ATCC 25922 (G-), Salmonella Enteritidis ATCC 13036(G-), Aspergillus niger ATCC 16404, Pennicilium citrinum ATCC 9849.

RESULTS AND DISCUSSION

A total of 70 volatile compounds were identified in the EO from C. citrates leaves (99.6 % of the total composition) (Table 1). As can be seen, oxygenated monoterpenes were the most represented class of volatiles with 86 %. Among them, neral and geranial were the most abundant. Monoterpene hydrocarbons were found as the second major chemical class (10.7%) with myrcene being the main component.

Table 1
Chemical composition (%) of the essential oil from C. citratus leaves.

Identity A identification based on the linear retention times (LRI) and mass spectra of pure compounds; B: identification based on LRI and mass spectra comparison with databases or literature data. tr: traces (< 0.1%).

The bioactivity of any plant species is attributed to its chemical compounds. If these constituents are secondary metabolites, they are often essential in many processes affecting plantgrowth, development, and environmental interaction. Many studies show that the chemical composition of EOs varies considerably according to several factors, including geobotanical conditions of the environment, cultivation method, plant age, photoperiod, harvest period, among others (Figueiredo et al., 2008). Nevertheless, our results are closely related with the chemical composition reported for the previously analyzed EO from the same region, where major constituents were geranial(41.3 %), neral (32.3 %) and myrcene (15.5 %) (Sacchetti et al., 2005) and for the EO from Burkina Faso were geranial (44.6 %), neral (33.0 %) and myrcene (10.7 %) (Menut et al., 2000). Other studies only found the citral isomers as major compounds: geranial (40.8 %) and neral (31.8 %) from plants grow in Manchester (UK) Tzortzakis & Economakis, (2007), geranial (31.7 %) and neral (46.2 %) (Leite et al., 2016) or geranial (29.2 %) and neral (27.0 %) (Athayde et al., 2016), both from Brazil.

Antioxidant properties of the EO were determined by two complementary methods: radical- scavenging capacity of the oil (ABTS) and ferric-reducing antioxidant power (FRAP) assay. The reducing effect on the radical cation ABTS was 26.8mmol/100 mL for the EO (Table 2). In the second assay, the FRAP were between 263.4 ± 13.3 to 404.3 ± 16.4 µmol/L of ascorbic acid equivalents by different concentrations. The chemical methods to evaluate the antioxidant capacity measure the combined effects of many substances present in the sample, which their antioxidant function is due to different mechanisms: elimination of free radicals generated in the assay (ABTS method) and the transfer of electrons in reactions of redox type (FRAP method). This explains the differences observed between the methods used, according to the results of the present study. Nevertheless, the weak activity is attributed to the presence of citral isomers (Baschieri et al., 2017). Unlike phenols, oxygenated monoterpenes exhibit less antioxidant activity (Sell, 2010), which would explain the lower antioxidant activity observed with respect to other EOs like oregano. It must be pointed out that EO from the present work performed worse than the EO from Ecuadorian- grown plants (Sacchetti et al., 2005) or of the same botanical source but of different geographical origin (Menut et al., 2000).

Table 2
Antioxidant effectiveness of the essential oil.

1 Antioxidant effectiveness expressed as ABTS and FRAP assays. Values represent an average of three determinations with standard deviation. 2nt: not tested

The MIC of the EO was ranged between 0.05-5 µL/mL (Table 3). The essential oil showed better activity against St. aureus and S. Enteritidis followed by E. coli. The activity was low against B. subtilis, A. niger and P. citrinum. In general, these results are in accordance with those reported for this EO (Raybudi-Massilia, Mosqueda-Melgar & Martín-Belloso, 2006; Tzortzakis & Economakis, 2007; Naik et al., 2010; Matasyoh et al., 2011; Leite et al., 2016; Athayde et al., 2016; Ekpenyong & Akpan, 2017) and they are attributed to the presence of citral isomers as responsible for showing antimicrobial activity against specific pathogens (Ekpenyong & Akpan, 2017; Abe et al., 2003).

Table 3
Minimal inhibitory concentrations of the essential oil against tested microorganisms.

CONCLUSIONS

The EO composition of Cympopogon citratus leaves show the presence of 70 volatile constituents, of which the most prominent were neral (33.2 %) and geranial (39.8 %). The EO had a low antioxidant activity by using the ABTSassay and it showed ferric reducing antioxidant power, while it has antimicrobial activity against St. aureus and S. enteritidis followed by E. coli. The activity was low against B. subtilis, A. niger and P. citrinum.

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Author notes