INTRODUCTION

Turmeric (Curcuma longa L.) is a rhizomatous herbaceous perennial plant of the Zingiberaceae family, native to tropical South Asia, but is now widely cultivated in the tropical and subtropical regions of the world (Remadevi et al., 2007). The deep orange- yellow powder known as turmeric is prepared from boiled and dried rhizomes of the plant. It has been commonly used as spice and medicine, particularly in Asia. The curcuminoid pigments and volatile oil, which are the major secondary metabolites of the rhizome, have been shown to be largely responsible for the pharmacological activities of turmeric powder, extracts and oleoresins (Remadevi et al., 2007; Sasikumar, 2001).

There are extensive in vitro and in vivo investigations on essential oil and extracts of turmeric extracts which showed hepato- and cardioprotective, hypoglycemic, anti-amyloidogenic, antifungal, parasiticidal, antioxidant, insect repelling, chemo-resistance and radio-resistance activities (Remadevi et al., 2007; Balakrishnan, 2007; Li et al., 2018). Although the fungicidal and bactericidal properties are traditionally known (Remadevi et al., 2007; Sasikumar, 2001; Balakrishnan, 2007; Li et al., 2018), reports about antifungal properties of the essential oil are scant and show a wide discrepancy about its inhibitory concentration in the substrate for the same fungal species (Saju et al., 1998; Behura et al., 2000; Jayaprakasha et al., 2001). This wide variation may be related to the chemical composition of the essential oil, which varies considerably among the cultivars, maturity stage, and cultivation prcedures (Singh et al., 2002; Ming et al., 2002; Raina et al., 2002; Pino et al., 2003; Singh et al., 2010; Liju et al., 2011; Gounder y Lingamallu, 2012; Avanço et al., 2017). In Ecuador, turmeric is commercially grown in the Amazonian region, where it is used as a food seasoning. However, only one study has been done about the chemical composition of its essential oil and biological activities (Sacchetti et al., 2005). Therefore, the present study was done to analyze the chemical composition and biological activities of the essential oil from turmeric (Curcuma longa L.) rhizomes grown in Amazonian Ecuador.

MATERIALS AND METHODS

Materials and isolation of essential oil

Rhizomes of C. longa were collected by Fundación 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. HERUTEQ1057). Fresh rhizomes (800 g) were steam distilled for 6 h in a pilot-scale distiller. The oil yield was 0.8% v/w.

Gas chromatography

Analyses of the essential oil 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 analysis parameters were: oven temperature program, 60 oC (2 min), 60–220 oC (4 oC/min) and 220 oC (5 min); hydrogen carrier gas flow rate 1 mL/min; injector and detector temperatures 250 oC. Samples (1 μL) were injected using split ratio 1:50, and previously diluted in n- pentane (1:6 v/v). The quantification of compounds was performed using relative percentage abundance and normalization method.

The essential oil was also examined by gas chromatography-mass spectrometry (GC-MS) using a QP-2010 Ultra (Shimadzu, Japan) with the same capillary column, temperature program and helium carrier gas flow rate 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 .-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 (Adams, 2001) 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.

Assay of 2,2-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity

The antioxidant activity of the essential oil was measured in terms of hydrogen-donating or radical scavenging ability, using the stable radical DPPH (Tabart, 2008). In the test tubes, 1.5 mL of DPPH (0.075 mg/mL) in ethanol was mixed with 750 μL of five concentrations of the essential oil to evaluate in a range of concentrations between 25-500 μg/mL. A control sample (absolute ethanol) and a reference sample (750 μL absolute ethanol and 1.5 mg/mL of DPPH solution) were also used. The decrease in the absorbance was determined at 515 nm, until the reaction plateau step was reached. Ascorbic acid was used as antioxidant standard. Three independent tests were performed for each sample. Then, the IC50 values (total antioxidant compound necessary to decrease the initial DPPH radical concentration by 50%) were determined.

Ferric-reducing antioxidant power (FRAP) assay

The FRAP of the essential oil was measured by the method reported earlier (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 essential oil (90 µL) was mixed with 900µL 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 4 min and the absorbance was measured at 593 nm. The FRAP was expressed in units of ascorbic acid equivalent.

Antimicrobial screening

For determination of minimal inhibitory concentration (MIC) 5, 0.5, 0.05 and 0.005 μL/mL of the essential oil were placed in different test tubes and 1 mL of dimethyl sulfoxide added to each of them. One milliliter of peptone water (Mueller Hinton broth) was added followed by addition of 1 mL of 24h–culture broth 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 and Pennicilium citrinum ATCC 9849.

RESULTS AND DISCUSSION

A total of 88 volatile compounds were identified in the essential oil from C. longa rhizomes (98.8% of the total composition) (Table 1). As can be seen, oxygenated sesquiterpenes were the most represented class of volatiles with 63.6 %. Among them, ar-turmerone and α- turmerone were the most abundant. Sesquiterpene hydrocarbons were found as the second major chemical class (18.0%) with α-zingiberene being the main component. Another individual major constituent was the monoterpene hydrocarbon α-phellandrene.

Table 1
Chemical composition (%) of the essential oil from C. longa rhizomes.

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 chemical composition of the turmeric rhizomes essential oil varies considerably according to several factors (Balakrishnan, 2007). These results disagree with those reported in the literature in both quality and quantity. The essential oil previously analyzed from the same region was dominated by α-phellandrene (20.4%), α-turmerone (19.8%), 1,8-cineole (10.3%), g-terpinene (6.1%) and β-turmerone (7.4%) (Sacchetti et al., 2005). The volatile oil obtained by hydrodistillation of turmeric rhizomes grown in the North Indian Plains contained 1,8-cineole (11.2%), α-turmerone (11.1%), β-caryophyllene (9.8%), ar-turmerone (7.3%) and β-sesquiphellandrene (7.1%) as major compounds (Raina et al., 2002), while the essential oil from Gorakhpur (India) was dominated by ar-turmerone (24.4%) and α- turmerone (20.5%) (Singh et al., 2010). The essential oil isolated from rhizomes grown in Kerala (India) was rich in ar-turmerone (61.8%), curlone (12.5%) and ar-curcumene (6.1%) (Liju et al., 2011); while that from Mysore (India) was dominated by ar-turmerone (21.0- 30.3%), α-turmerone (26.5-33.5%) and β-turmerone (18.9 - 21.1 %) (Gounder y Lingamallu, 2012). The main components of the essential oil from Brazil were α-turmerone (42.6%), β- turmerone (16.0 %) and ar-turmerone (12.9%) (Avanço et al., 2017); while the composition of the essential oil from Cuban rhizomes was close to those found in the present work: ar- turmerone (47.7%) and α-turmerone (16.1%) (Pino et al., 2003).

Antioxidant properties of the essential oil were determined by two methods: radical- scavenging capacity of the oil (or DPPH bleaching) and ferric-reducing antioxidant power (FRAP) assay (Table 2). The IC50 value was14.5 mg/mL for the essential oil with a maximal effect of 30 % at 60 mg/mL. The radical scavenging potential of turmeric essential oil was much lower when compared to those of standard ascorbic acid and Trolox. This performance was lower than those reported previously for a sample obtained from the same region (Sacchetti et al., 2005) and a sample from Mysore (India) (Gounder y Lingamallu, 2012).

Table 2
Antioxidant effectiveness of the essential oil

1 Antioxidant effectiveness expressed as IC50 (DPPH scavenger activity) and FRAP (in units of ascorbic acid equivalent). Values represent an average of three determinations with standard deviation.

In the second assay, at low pH ferric tripyridyltriazine (Fe³⁺-TPTZ) complex is reduced to ferrous [Fe²⁺] form, an intense blue colored complex with absorption maximum at 593 nm by the electron donating action of antioxidant (Benzie, 1996). The FRAP was 389.0 ± 12.0µM of ascorbic acid equivalents and the essential oil showed less activity than Trolox. Probably, the sesquiterpene ketones ar-turmerone and α-turmerone exert either the synergistic or additive actions towards the total antioxidant activity. It may also be possible that these compounds alone or in synergy with other compounds present in the essential oil are responsible for the observed antioxidant activity of rhizomes of C. longa (Singh et al., 2010).

The MIC of the essential oil was ranged between 0.05-5 µL/mL (Table 3). The essential oil showed better activity against St. aureus and B. subtilis followed by P. citrinum. As it is commonly found, Gram+ bacteria were more susceptible to the essential oil than Gram- ones (Kalemba y Kunicka, 2003). In general, these results are in accordance with those reported for this essential oil (Singh et al., 2002; Sacchetti et al., 2005; Singh et al., 2011) and they are attributed to the presence of tumerones as responsible for showing antimicrobial activity against specific pathogens (Dhingra et al., 2007; Singh et al., 2011).

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

No inhibition.

CONCLUSIONS

Essential oil composition of Curcuma longa rhizomes show the presence of 88 volatile constituents, of which the most prominent were ar-turmerone (45.5%) and α-turmerone (13.4%). The essential oil had a low antioxidant activity by using the DPPH assay and it showed ferric reducing antioxidant power, while it has antimicrobial activity against gram positive bacteria (Bacillus subtilis and Staphylococcus aureus) and Pennicilium citrinum. No antimicrobial activity was found against Escherichia coli, Salmonella Enteritidis and Aspergillus niger.

REFERENCES

Adams, R. P. (2001). Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy. Carol Stream, IL: Allured Publishing Co.

Avanço, G. B., Ferreira, F. D., Bonfim, N. S., dos Santos, P. A. d. S. R., Peralta, R. M., Brugnari, T., Mallmann, C. A., de Abreu Filho, B. A., Mikcha, J. M. G., & Machinski, M. Jr. (2017). et al. Curcuma longa L. essential oil composition, antioxidant effect, and effect on Fusarium verticillioides and fumonisin production. Food Control, 73, 806-813.

Balakrishnan, K. V. (2007). Postharvest technology and processing of turmeric. En: P. N. Ravindran, K. Nirmal Babu, & K. Sivaraman (Eds.), Turmeric: the genus Curcuma (pp. 193-256). Boca Raton: CRC Press.

Behura, C., Ray, P., Rath, C. C., Mishra, R. K., Ramachandraiah, O. S., & Charyulu, J. K. (2000). Antifungal activity of essential oils of Curcuma longa against five rice pathogens in vitro. J. Essent. Oil Bear. Plants, 3, 79-84.

Benzie, I. F.F. (1996). An automated, specific, spectrophotometric method for measuring ascorbic acid in plasma (EFTSA). Clinical Biochem., 29, 111-116.

Dhingra, O. D., Jham, G. N., Barcelos, R. C., Mendonça, F. A., & Ghiviriga, I. (2007). Isolation and identification of the principal fungitoxic component of turmeric essential oil. J. Essent. Oil Res., 19(4), 387-391.

Gounder, D. K., & Lingamallu, J. (2012). Comparison of chemical composition and antioxidant potential of volatile oil from fresh, dried and cured turmeric (Curcuma longa) rhizomes. Ind. Crops Prod., 38, 124-131.

Jayaprakasha, G. K., Negi, P. S., Anandharamakrishnan, C., & Sakariah, K. (2001). Chemical composition of turmeric oil- a byproduct of oleoresin industry and its inhibitory activity against different fungi. Z. Naturforschung, 56c, 40-44.

Kalemba, D., & Kunicka, A. (2003). Antibacterial and antifungal properties of essential oils. Curr. Medicinal Chem., 10, 813-29.

Li, S., Yuan, W., Deng, G., Wang, P., Yang, P., & Aggarwal, B. (2011). Chemical composition and product quality control of turmeric (Curcuma longa L.). Faculty Publications. Paper 1.http://scholarworks.sfasu.edu/agriculture_facultypubs/1. Downloaded February 15, 2018.

Liju, V. B., Jeena, K., & Kuttan, R. (2011). An evaluation of antioxidant, anti- inflammatory, and antinociceptive activities of essential oil from Curcuma longa L. Indian J. Pharmacol., 43(5), 526-532.

Ming, J. C., Vera, R., Chalchat, J. C., & Cabassu P. (2002). Chemical composition of essential oils from rhizomes, leaves, and flowers of Curcuma longa L. from Reunion Island. J. Essent. Oil Res., 14, 249-251.

Pino, J., Marbot, R., Palau, E., & Roncal, E. (2003). Essential oil constituents from Cuban turmeric rhizomes. Rev. Lat. Quim., 31, 16-19.

Raina, V. K., Srivastava, S. K., Jain, N., Ahmad, A., SyMsundar, K. V., & Aggarwal, K. K. (2002). Essential oil composition of Curcuma longa L. cv. Roma from plains of northern India. Flavour Fragr J., 17, 99-102.

Remadevi, R., Surendran, E., & Kimura, T. (2007). Turmeric in traditional medicine. En: P. N. Ravindran, K. Nirmal Babu, & K. Sivaraman (Eds.), Turmeric: the genus Curcuma (pp. 409-436). Boca Raton: CRC Press.

Sacchetti, G., Maietti, S., Muzzoli, M., Scaglianti, M., Manfredini, S., Radice, M., & Bruni, R. (2005). Comparative evaluation of 11 essential oils of different origin as functional antioxidants, antiradicals and antimicrobials in foods. Food Chem., 91, 621- 632.

Saju, K. A., Venugopal, M. N., & Mathew M. J. (1998). Antifungal and insect repellent activities of essential oil of turmeric (Curcuma longa L.). Current Sci., 75, 660-662.

Sasikumar. B. (2001). Turmeric. En: K. V. Peter (Ed.), Handbook of Herbs and Spices (pp. 297-310). Cambridge: Woodhead Publishing Limited.

Singh, G., Kapoor, I. P. S., Singh, P., de Heluani, C. S., de Lampasona, M. P., & Catalan, C. A. N. (2010). Comparative study of chemical composition and antioxidant activity of fresh and dry rhizomes of turmeric (Curcuma longa Linn.). Food Chem. Toxicol., 48, 1026-31.

Singh, R., Chandra, R., Bose, M., & Luthra, P. M. (2002). Antibacterial activity of Curcuma longa rhizome extract on pathogenic bacteria. Current Sci., 83, 737-740.

Singh, S., Sankar, B., Rajesh, S., Sahoo, K., Subudhi, E., & Nayak, S. (2011). Chemical composition of turmeric oil (Curcuma longa L. cv. Roma) and its antimicrobial activity against eye infecting pathogens. J. Essent. Oil Res., 23(6), 11-18.

Tabart, J. (2008). Comparative antioxidant capacities of phenolic compounds measured by various test. Food Chem., 40, 123-128.

Author notes