ANTIOXIDANT ACTIVITY OF TAGETES ERECTA ESSENTIAL OIL

ROSA MARTHA PÉREZ GUTIÉRREZ, HELIODORO HERNÁNDEZ LUNA, SERGIO HERNÁNDEZ GARRIDO

Laboratorio de Investigación de Productos Naturales. Escuela Superior de Ingeniería Química e Industrias extractivas IPN. México D.F. México.

ABSTRACT

The essential oil from flowers of Tagetes erecta was evaluated for antioxidant activity in vitro using diphenyl-1-picrylhydrazyl (DPPH), thiocyanate, b-carotene bleaching, free radical scavenging activity and oxidation of deoxyribose assay. The GC-MS analysis of the oil has resulted in the identification of 18 components; b-caryophyllene, limonene, methyleugenol, (E)-ocimene, piperetone, piperitenone and ?-terpinolene were the main components.

Keywords: Tagetes erecta, essential oil, antioxidant activity

INTODUCTION

Plant essential oils as antioxidants have a protective role for highly unsaturated lipids in animal tissues (1). The oils have shown their action as those of hepatoprotective agents in ageing mammals in particular the long chain C20 and C22 acids. In addition, volatile oils also demonstrated a positive effect upon docosahexaenoic acid (DHA) levels in ageing rodent retinas. The reason that antioxidants are important to human physical well being comes from the fact that oxygen is a potentially toxic element since it can be transformed by metabolic activity into more reactive forms such as superoxide, hydrogen peroxide, singlet oxygen and hydroxyl radicals, collectively known as active oxygen. These molecules are formed in living cells by various metabolic pathways. Several substances have been proposed to act as antioxidants in vivo. They include b-carotene, albumin, uric acid, oestrogens, polyamines, flavonoids, ascorbic acid, plant phenolics, vitamin E and some drugs such as non-steroidal anti-inflammatories. They can stabilise the membranes by decreasing their permeability and they also have an ability to bind free fatty acids. It has been suggested that volatile oils could act as such agents. It has been found that certain volatile oils and their components are cytostatic to tumour cell lines and can offer potential as novel antiproliferative agents.

Tagetes erecta L. (Compositae) is the Aztec Marigold, native to Mexico is commonly know as Cempasuchil, Cempoalxóchitl or Flor de muerto, not to be confused with calendula. An annual herb whose flowerheads and foliage are used for their vermifugal properties, to treat colic and as an emmenagogue. The flowers are used in ornamental ofrends to celebrate in México religious day "día de muertos". The essential oil is characterized by a bitter, green, herbaceous  odor, the penetrating aroma has the curious property of becoming stronger when combined with other essences. It is used in the treatment of fungal infections and is employed in some herbaceous and floral perfumes and to deter house flies. Excessive skin use is discouraged as it can cause serious photosensitivity; do not use prior to exposure to simulated or natural sunlight. Essential oil of leaf completely inhibited the growth of Fusarium oxysporum and Trichophyton mentagrophytes and the mycelial growth of Pythium aphanidermatatum (2,3). When tested against 40 strains of bacteria and fungi, essential oil of Tagetes was found to have a 100% inhibitory effect against Gram-positive bacteria and fungi, and a 95% inhibitory effect against fungi (4). In this paper, we had report the antioxidant activity of essential oil of Tagetes erecta.

MATERIAL AND METHODS

Plant material. The material used in studies for antioxidant activity were purchased from markets in México D.F. The material was identified in the Department of Botany of ENEP-Iztacala UNAM, and a voucher specimen of the plant (5265) is stored in the herbarium of this Department for reference.

Extraction of the oil. Flowers were continuously extracted with light petroleum (bp. 40-60°C) using a Soxhlet apparatus. The solvent was removed under vacuum. Extraction of the brownish residue was with steam distilled, gave the volatile oil.

GC-MS analysis. Analyses were carried out on a Hewlett-Packard 5890 Series II Plus gas chromatograph interfaced to a Hewlett Packard 5989B mass spectrometer. Separations were performed on Ultra 1 (49 m x 0.20 mm I.D., 0.11 mm, Hewlett-Packard) and DB-Wax (60 m x 0.25 mm 1.0., 0.25 mm, J&W Scientific) capillary columns. Nitrogen was used as a carrier gas (1.0 ml/min C.F.) and the oven temperature was programmed as 60° to 230°C with a heating rate of 2°C/min. Injector and interface temperatures were 230°C and 255°C, respectively. El mass spectra were recorded at 70 eV ionization voltage over the mass range 40-400 u. Samples (0.5 mL of oil solutions 1:10 in hexane) were injected by split injection. Identification of the compounds was performed by aid of the computer library search (CAS No. 5989-27-5, Entry 8747, LIB#1). using a mass spectra library.

Antioxidant Activity Evaluation

Rapid screening for antioxidants. The oil (0.5 mL, 1:10 in chloroform) was tested for its antioxidant activity starting with a rapid TLC screening. In the first test the TLC sheet with a solution of oil was sprayed. The chromatogram was development in CH₂Cl₂:CH₃-CO-CH ₃ (25:1), and exposed to daylight until the background colors was bleached (45 min after spraying) zones in which a yellow colors persisted possessed antioxidant activity. After were sprayed with b-carotene-linoleic acid reagent and 0.2% solution of the stable radical diphenylpicrylhydrazyl, DPPH (5,6). The DPPH reagent the compounds were detected as yellow spots on a violet background. Only zones where the color turned from purple to yellow within the first 30 min (after spraying) were taken as positive results. The oil was positive and were subjected to further testing.

b-carotene bleaching method. The test was carried out following the spectrophotometric method of Miller, (7) based on the ability to decrease the oxidative bleaching on b-carotene in a b-carotene/linoleic acid emulsion. A 2.0, 4.0 and 6.0 mg sample of crystalline b-carotene was dissolved in 10 mL of chloroform, and 1 mL of this solution was pipetted into a round-bottom flask containing 20 mg of linoleic acid and 200 mg of Tween 40. After the removal of chloroform by evaporation, 50 mL of distilled water was added to the flask under vigorous stirring. After, 5 mL of the mixture were pipetted into test tubes containing 0.2 mL of the sample (100 mL), and the mixture was mixed wheel. For one sample the absorbance at 470 nm was immediately measured using the spectrophotometer, and for the other samples absorbance was measured after 10, 20, 30 and 90 min of incubation in a water bath at 50 C. Each sample was read against an emulsion prepared as described but without b-carotene (blank). To correct for the influence of the oil color in calculating b-carotene degradation rate, four aliquots (50 mL) of each sample were added to 5 mL of blank (blank sample), (8). The absorbances of the mixtures for each time point were read with a spectrophotometer, and the absorbance measured was subtracted from that of the corresponding sample. The degradation rate of b-carotene was calculated by first-order kinetics:

[ln(A₀/A_t)]/t = degradation rate (dr) of sample

Where A₀ = absorbance of the sample the absorbance of blank sample at time 0 (absorbance was read immediately after the addition of alkaloids solutions).

A_t = absorbance of the sample- absorbance of blank sample at time t, and t=10 or 20 or 30 or 90 min of incubation at 50 °C.

[ln(A₀/A_t)]/t = degradation rate (dr) of control sample

50 µL of sample was added to 5 mL of b-carotene emulsion and treated as the corresponding sample, A₀ = absorbance of the control sample at time 0, and A_t = absorbance of the control sample at time t. Antioxidant activity was expressed as the percent of inhibition relative to the control using the equation:

Thiocyanate Assays. Different amounts of samples dissolved in 120 mL of EtOH were added to a reaction mixture in a screw cap vial. Each reaction mixture consisted of 2.88 µL of 2.51% linoleic acid in EtOH and 9 ml of 40 mM phosphate buffer (pH 7.0). The vial was placed in a oven at 40 °C in the dark. At intervals during incubation, a 0.111 mL aliquot of the mixture was diluted with 9.7 mL of 75% EtOH, which was followed by adding 0.1 mL of 30% ammonium thiocyanate. Precisely 3 min after the addition of 0.1 mL of 20mM ferrous chloride in 3.5% hydrochloric acid to the reaction mixture, the absorbance of red color was measured at 500 nm every 24 h. All tests were run in triplicate (9). In the antioxidation assay the following antioxidants were tested with a-tocopherol.

Free Radical Scavenging Activity

The stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging effect of oil were carried out as described before (10,11). Oil were mixed with 0.1 mM DPPH radical in ethanol solution. A 20 min incubation period at room temperature was used before reading the absorbance at 519 nm. All the test and analyses were performed in triplicate and average. The inhibitory percentage of DPPH was calculated according to the following equation:

Deoxyribose Assay. In this Assay, a Fenton reaction (13,14) based model ABTS (ferrimioglobin/2,2´-azino-bis-(3-ethylbenztiazoline-6-sul fonic acid) radical cation model was also used to evaluate the free radical scavenging effect of oil by compared by determining the percentage of decolorization at 734 nm after 8 min of reaction at room temperature (12). The effect of oil on scavenging ABTS radical was calculated from the following equation:

Deoxyribose Assay. In this Assay, a Fenton reaction (13,14) based model containing 0.1 mM of Fe³⁺ or Cu²⁺. All solutions were prepared freshly, 1.0 mL of the reaction contained 100 mM of 28 mM 2-deoxy-2-ribose (Fluka, dissolved in KH₂PO₄-K₂HPO₄ buffer pH 7.4), 500 mL solution of various concentrations of the oil (in buffer), 200 mL of 200 mM FeCl₃ and 1.04 µM EDTA (1:1 v/v), 100 mL H₂O₂ (1.0 mM) and 100 mL ascorbic acid (1.0 µM). After an incubation period of 1 h at 37 °C the extent of deoxyribose degradation was measured by the TBA reaction. 1.0 mL of TBA (1% in 50 mM NaOH) and 1.0 mL of TCA were added to the reaction mixture and the tubes were heated at 100 °C for 20 min. After cooling the absorbance was read at 532 nm against a blank (containing only buffer and deoxyribose). The absorbance (A1) read at the end of the experiment was used for the calculation of the percentage inhibition of deoxyribose degradation by the test compound.

Calculation were done as:

I(%)= 100 X (A₀ - A₁/A₀)

A₀ = Was the absorbance of the control reaction (full reaction, containing no test compounds) and A₁= was the absorbance in the presence of the inhibitor.

The IC₅₀ value represented the concentration of the compounds, that caused 50% inhibition. All experiments were carried out in triplicated.

RESULTS AND DISCUSION

The essential oil of T. erecta which was obtained by Soxhlet extraction and hydrodistillation of the fresh flowers. The percentage of the essential oil was of 0.4%. GC-MS analysis resulted in the identification of 18 compounds (Table 1). Among these b-caryophyllene (15.2%), limonene (11.7%), methyleugenol (12.3%), (E)-ocimene (13.7%), piperetone (19.2%), piperitenone (8.1%) and a-terpinolene (11.9%) being the main components. While the other components were present as minors or traces. Only quantitative and not qualitative variations were found in the oil T. erecta collected in México of the other ornamental variants find for other countries (15-18).

b-carotene bleaching method. The Table 2 shows the decrease in absorbance during the coupled oxidation of b-carotene and linoleic acid. The b-carotene bleaching test was selected for antioxidant activity determination because it is carried out in an emulsion, a situation frequent in foods. On the other hand, it is generally agreed that the oxidation is initiated by free radical attack; therefore, assays to evaluate the radical scavenging activity are representative of the potential of a compound to retard oxidation. Among the radical scavenging assays, the one based on the utilization of DPPH was chosen due to its simplicity and worldwide acceptance for comparative purposes (7).

Effect of oil on the oxidation of linoleic acid. The effect of oil on the oxidation of linoleic acid, determined by the thiocyanate method, are in Table 3. The autoxidation of linoleic acid without adding oil and antioxidant (a-tocoferol) was accompanied by a rapid increase of peroxide value. Significant differences (P< 0.05) in peroxide value were found between the control and the linoleic acid containing oil or antioxidant. The antioxidative activity of the oil increased with concentration up to 12.5 µg/mL and decreased at higher concentrations of oil, that is, 200 mg/mL. This might be explained by the fact that at higher concentrations oil acts as oxygen-carrying agent (19) and serves as a pro-oxidant in the co-oxidation of linoleic acid. The thiocyanate test showed that at a concentration of 50 mg/mL the inhibition of oxidation of linoleic acid produced by oil was 71.1 % respectively as compared to the blank, was less efficient, whereas the antioxidant efficiency of a- tocopherol was of 92.1 %.

DPPH assay. The DPPH method is considered to be a model compound of a lipophilic radical. Lipid autoxidation initiates a chain reaction in lipophilic radical. The scavenging effects of essential oil on DPPH radical are shown in Table 4, were found to have lower scavenging effects. Oil exhibited antioxidative activities against lipid peroxidation and peroxyl radical. In the ABTS (ferrimioglobin/2,2´-azino-bis-(3-ethylbenztiazoline-6-sulfonic acid) model reaction (Table 5), exhibited the inhibitory activity in the ABTS radical at 100 mg (57.3 %) less than a-tocoferol (70.5%).

Effect of oil on the oxidation of deoxyribose. The effect of oil on deoxyribose damage induced by Fe³⁺/H₂O₂ is shown in Table 6. The at 100 mg/mL showed 73.6%, less than a-tocoferol (94.2%). If a Fe²⁺- EDTA chelate was incubated with deoxyribose in phosphate buffer at pH 7.4 ^.OH radicals are formed (20). Any such radical that escaped scavenging by the EDTA itself carreact with deoxyribose. The rate of deoxyribose degradation may be increased by ascorbic acid and H₂O₂. The oil is capable of compete with deoxyribose for ^.OH, it will decrease the rate of deoxyribose degradation (21). Oil showed a protective effect from oxidative damage of deoxyribose. The ability to reduce Fe³⁺ and stimulate deoxyribose degradation has been adopted as one measure of pro-oxidant properties and can applied to study a variety of food additives (22).

In conclusion, our results demonstrate that essential oil from Tagetes erecta at the dose level proved possesses significant antioxidant activity less than a-tocoferol.

The antioxidant activity of the essential oil from Tagetes erecta found in the present study may be due to the presence of the camphor and methyleugenol. Both are a chemical compounds occurring naturally in a variety of spices and herbs it are used as a flavoring agent. Previous studies demonstrated that camphor (23) and methyleugenol (24) possess antioxidant activity.

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