Chemical Study and Antimalarial,Antioxidant,and Anticancer Activities of Melaleuca armillaris (Sol Ex Gateau) Sm Essential Oil

Abstract

This study investigated the chemical composition (by using gas chromatography/flame ionization detection and gas chromatography/mass spectrometry, an antioxidant [1,1-diphenyl-2-picrylhydrazyl] [DPPH] radical–scavenging assay, and a 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate [ABTS] radical cation–scavenging assay) and the antimalarial and cytotoxic activities of essential oil extracted from leaves of Melaleuca armillaris. Thirty-two components representing more than 98% of the total composition of the essential oil were identified. The main components were 1,8-cineole (85.8%), camphene (5.05%), and α-pinene (1.95%). The antioxidant activity by ABTS assay showed a mean (±standard deviation) 50% inhibitory concentration (IC₅₀) value of 247.3±3.9 mg/L, and the DPPH assay yielded an IC₅₀ value of 2183.6±44.3 mg/L. The antimalarial study indicated that the essential oil had mild activity against the chloroquine-resistant Plasmodium falciparum FcB1 strain (IC₅₀, 27±2 mg/L). The cytotoxic activity of this essential oil was tested against MCF7 human breast cancer cells and was found to be high (IC₅₀, 12±1 mg/L).

Introduction

I n developing countries, large segments of the population still rely on traditional medicine to treat serious diseases, such as infections, cancer, and different types of inflammatory diseases. Thus, natural products, including medicinal plants, have clearly become an important source from which to extract and identify new molecules (or at least new lead structures) for the development of new drugs for treatment of these diseases.¹ For example, essential oils extracted from aromatic and medicinal plants are widely used in pharmaceutical industries. They have been known since antiquity for their biological activities, notably as antibacterial, antifungal, or antioxidant agents. With the growing interest in the use of essential oils in both food and pharmaceuticals, a systematic examination of plant extracts for these properties has become more and more important.² Consumers have increasingly expressed an interest in limiting the use of synthetic drugs because those chemicals can be a potential health hazard as a result of toxic impurities derived from the synthetic pathway.³

The genus Melaleuca, with about 250 species, is one of the most widespread members of Myrtaceae family. It is native of Australia and nearby islands. This large genus approaches Eucalyptus in both abundance and diversity.⁴ Some Melaleuca species are rich in essential oils and are used for medicinal purposes, against insects, and in cosmetic preparations.⁵ However, M. alternifolia is the main commercial source of the essential oil known as melaleuca oil.⁶ This term has been selected as the approved official name by the Therapeutic Goods Administration of Australia. The essential oil is a Melaleuca byproduct that has attracted keen interest. In recent years, the Melaleuca essential oil has been identified in different parts of the plant. Terpinolene, 1,8-cineole, α-pinene, β-pinene, terpinen-4-ol, and other elements are the major aromatic compounds of many Melaleuca species.^4,7

M. armillaris (Sol Ex Gateau) Sm is called the bracelet honey myrtle. The essential oil possesses antimicrobial, antifungal, fumigant, and antioxidant activities.^8,9 Given the medicinal interest in the species, several studies examined the chemical characterization of the essential oil. However, these studies were mostly related to M. alternifolia. Little information is available about the chemical composition or the biological activities of M. armillaris. To our knowledge, no reports in the literature describe any cytotoxic, antimalarial, or antioxidant (according to the 1,1-diphenyl-2-picrylhydrazyl [DPPH] or 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate [ABTS] assays) activities for M. armillaris essential oil. Therefore, we sought to analyze the chemical composition of hydrodistilled essential oil of M. armillaris by gas chromatography/flame ionization detection (GC-FID) and gas chromatography/mass spectrometry (GC-MS); to determine essential oil chemotypes; and to investigate the antioxidant, antimalarial, and cytotoxic activities of these chemotypes.

Materials and Methods

Plant material

The leaves of M. armillaris were collected in January 2008 from the area of Tunis, Tunisia. Specimens were identified by Dr. Nadia Ben Brahim at the Department of Botany, National Institute of Agronomic Research in Tunis, and voucher specimens were deposited at the Herbarium of the Department of Botany in the cited institute.

Extraction of the essential oil

The oil of dried samples of M. armillaris leaves was extracted by using a Clevenger-type apparatus according to the method recommended by the European Pharmacopoeia.¹⁰ The aerial parts of the plant were air-dried at room temperature. The hydrodistillation was performed with 30 g of dried leaves and 360 mL of pure water. The distilled essential oil was dried over anhydrous sodium sulfate, filtered, and stored in sealed vials at 4°C before further analyses.

Chemicals used

All chemicals used were of analytical reagent grade. All reagents were purchased from Sigma, Aldrich, Fluka (Saint-Quentin, France).

Antioxidant activity

Free radical–scavenging activity: DPPH test

Antioxidant scavenging activity was studied by using DPPH as described by Blois,¹¹ with some modifications. A total of 1.5 mL of various dilutions of the test materials (pure antioxidant or essential oil) were mixed with 1.5 mL of a 0.2-mmol/L methanolic DPPH solution. After an incubation period of 30 minutes at 25°C, the absorbance at 520 nm (the wavelength of maximum absorbance of DPPH) was recorded as A_(sample) using a Helios spectrophotometer (Unicam, Cambridge, United Kingdom). A blank experiment was also carried out using the same procedure to a solution without the test material; the absorbance was recorded as A_(blank). The free radical–scavenging activity of each solution was then calculated as the percentage inhibition according to the following equation:

\documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*} \% {\rm inhibition} = 100 ({\rm A}_{({\rm blank})} -{\rm A}_{({\rm sample})})/{\rm A}_{({\rm blank})} \end{align*} \end{document}

Antioxidant activities of test compounds or the essential oil were expressed as IC₅₀, defined as the concentration of the test material required to cause a 50% decrease in initial DPPH concentration. Ascorbic acid was used as a standard. All measurements were performed in triplicate.

ABTS radical-scavenging assay

The radical-scavenging capacity of the samples for the ABTS radical cation was determined as described by Re et al.,¹² with some modifications. ABTS was generated by mixing 7 mmol/L of ABTS at a pH of 7.4 (5 mmol/L NaH₂PO₄, 5 mmol/L Na₂HPO₄, and 154 mmol/L NaCl) with 2.5 mmol/L potassium persulfate (final concentration), followed by storage in the dark at room temperature for 16 hours before use. The mixture was diluted with ethanol to give an absorbance of 0.70±0.02 units at 734 nm using a spectrophotometer (Helios). For the sample, solutions of the essential oil in methanol (100 μL) were allowed to react with fresh ABTS solution (900 μL); the absorbance was then measured 6 minutes after initial mixing. Ascorbic acid was used as a standard and the capacity of free-radical scavenging was expressed as IC₅₀ (mg/L). IC₅₀ values were calculated as the concentration required to scavenge 50% of ABTS radicals. The capacity of free radical–scavenging IC₅₀ was determined by using the same previously used equation for the DPPH method. All measurements were performed in triplicate.

All data on antioxidant activity were expressed as means±standard deviations (SDs) of triplicate measurements. The confidence limits were set at a P value less than .05. SDs did not exceed 5% for the majority of the values obtained.

GC and GC-MS

Quantitative and qualitative analyses of the essential oil were carried out by GC-FID and GC-MS. GC analyses were performed on a Varian Star 3400 (Les Ulis, France) Cx chromatograph fitted with a fused silica capillary DB-5MS column (5% phenylmethylpolysyloxane, 30 m×0.25 mm, film thickness 0.25 μm). Chromatographic conditions were a temperature increase from 60°C to 260°C, with a gradient of 5°C/min and 15 minutes isotherm at 260°C. A second gradient was applied to 340°C at 40°C/min. Total analysis time was 57 minutes.

For analysis, essential oil was dissolved in petroleum ether. One microliter of sample was injected in the split-mode ratio of 1:10. Helium (purity, 99.999%) was used as carrier gas at 1 mL/min. The injector was operated at 200°C. The mass spectrometer (Varian Saturn GC/MS/MS 4D) was adjusted for an emission current of 10 μA and electron multiplier voltage between 1400 and 1500 V. Trap temperature was 150°C and that of the transfer line was 170°C. Mass scanning was from 40 to 650 amu.

Compounds were identified by comparison of their Kovats indices relative to C5-C24 n-alkanes obtained on a nonpolar DB-5MS column with those provided in the literature, by comparison of their mass spectra with those recorded in NIST 08 (National Institute of Standards and Technology) and reported in published articles, and by co-injection of available reference compounds. The samples were analyzed in duplicate.

The percentage composition of the essential oil was computed by the normalization method from the GC peak areas, assuming identical mass response factor for all compounds. Results were calculated as mean values of 2 injections from essential oil, without using correction factors. All determinations were performed in triplicate and averaged.

Antiplasmodial activity

The chloroquine-resistant FcB1-Columbia strain of Plasmodium falciparum (IC₅₀ for chloroquine, 186 nmol/L) was cultured continuously according to Trager and Jensen¹³ with modifications described by Benoit et al.¹⁴ The IC₅₀ values for chloroquine were checked every 2 months, and we observed no significant variations. The parasites were maintained in vitro in human red blood cells (O±; EFS; Toulouse, France), diluted to 4% hematocrit in Roswell Park Memorial Institute medium 1640 (Lonza; Emerainville, France) supplemented with 25 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 30 M NaHCO₃ and complemented with 7% human AB+ serum (EFS).

Parasite cultures were synchronized by combination of magnetic enrichment¹⁵ and by D-sorbitol lysis (5% of D-sorbitol in sterile water) as described by Lambros and Vanderberg.¹⁶ The antimalarial activity of essential oil was evaluated by a radioactive micromethod described elsewhere.¹⁷ Tests were performed in triplicate in 96-well culture plates (TPP) with cultures mostly at ring stages (synchronization interval, 16 hours) at 0.5%–1% parasitemia (hematocrit, 1.5%). Parasite culture was incubated with each sample for 48 hours. Parasite growth was estimated by (³H)-hypoxanthine (Perkin-Elmer; Courtaboeuf, France) incorporation, which was added to the plates 24 hours before freezing. After 48-hour incubation, plates were frozen-defrosted and each well was harvested on a glass fiber filter. Incorporated (³H)-hypoxanthine was then determined with a beta-counter (1450-Microbeta Trilux; Wallac-Perkin Elmer). The control parasite cultures, free from any sample, were referred to as 100% growth. IC₅₀ values were determined graphically in concentration versus percentage inhibition curves. Chloroquine diphosphate was used as a positive control.

The antimalarial activity of samples was expressed by IC₅₀, representing the concentration of drug that induced a 50% parasitemia decrease compared with the positive control culture (referred to as 100% parasitemia). According to the literature on plant antiplasmodial activities a sample is very active if the IC₅₀ is less than 5 mg/L, active if the IC₅₀ was 5–50 mg/L, weakly active if the IC₅₀ was 50–100 mg/L, and inactive if the IC₅₀ was greater than 100 mg/L.¹⁸

Cytotoxicity evaluation

Cytotoxicity of the sample was estimated on human breast cancer cells (MCF7). The cells were cultured in the same conditions as those used for P. falciparum, except for the 10% human serum, which was replaced by 10% fetal calf serum (Lonza). For the determination of pure compound activity, cells were distributed in 96-well plates at 3×10⁴ cells/well in 100 μL; 100 μL of culture medium containing sample at various concentrations was then added. Cell growth was estimated by incorporating (³H)-hypoxanthine after 48-hours incubation, exactly as was done for the P. falciparum assay. The (³H)-hypoxanthine incorporation in the presence of sample was compared with that of control cultures without sample (the positive control was doxorubicin).¹⁹

Results and Discussion

Chemical analysis of the essential oil

The hydrodistillation of dried M. armillaris leaves yielded yellowish essential oil (yields of 4.28%). This yield was close to results recorded for M. armillaris in Australia (yields of 4.66%).⁹

Table 1 shows the qualitative and quantitative composition of the essential oil. The GC-MS analysis of the essential oil led to the identification of 32 different components, representing 98.69% of the total oil constituents. The essential oil contained a complex mixture consisting mainly of oxygenated monoterpene (86.51%), monoterpene hydrocarbons (10.33%), sesquiterpene hydrocarbons (1.05%), and oxygenated sesquiterpene (0.77%). The major monoterpenes identified in the essential oil of M. armillaris leaves were 1,8-cineole (85.80%), camphene (5.05%), α-pinene (1.95%), and sabinene (1.30%) (Fig. 1). The percentage and compositions of essential oil of M. armillaris leaves were significantly different from those of the essential oil obtained from leaves of Australian origin.⁹ The Australian essential oil contained major components: 1,8-cineole (42.77%), α-thujene (2.07%), α-pinene (2.52%), sabinene (3.55%), limonene (4.54%), γ-terpinene (8.86%), terpinen-4-ol (15.97%), and α-terpineol (4.56%). Farag et al.⁸ reported that the essential oil of M. armillaris contained 1,8-cineole (33.93%) and terpinen-4-ol (18.79%). Hayouni et al.²⁰ reported the following major components: 1,8-cineole (68.9%), borneol (6.16%), α-pinene (2.94%), and β-myrcene (2.79%).

FIG. 1.

Structures of abundant compounds identified in Melaleuca armillaris essential oil.

Table 1.

Chemical Composition of Essential Oil Extracted from Melaleuca armillaris leaves

	Kovats Index	Compound	% Area	Components (%)
1	866	m-xylene	0.03
2	930	α-thujene	0.06
3	936	α-pinene	1.95
4	951	α-fenchene	0.02
5	954	Camphene	5.05
6	974	β-pinene	0.33
7	974	Sabinene	1.30
8	985	β-myrcene	0.25
9	1011	δ-3-carene	0.44
10	1025	p-cymene	0.13
11	1030	1,8-cineole	85.80
12	1030	α-phellandrene	0.13
13	1033	β-phellandrene	0.07
14	1057	γ-terpinene	0.04
15	1086	α-terpinolene	0.56
16	1094	Linalool	0.04
17	1166	Borneol	0.38
18	1185	α-terpineol	0.16
19	1389	β-elemene	0.26
20	1415	β-caryophyllene	0.05
21	1450	α-himachalene	0.05
22	1480	γ-muurolene	0.02
23	1482	β-selinene	0.12
24	1488	α-amorphene	0.05
25	1513	γ-cadinene	0.12
26	1524	δ-cadinene	0.36
27	1550	Elemol	0.09
28	1554	Germacrene B	0.02
29	1580	Caryophyllene oxide	0.44
30	1638	Spathulenol	0.22
31	1654	α-cadinol	0.02
32	ND	p-menth-4(8)-en-9-ol^a	0.13
Identified components				98.69
Monoterpene hydrocarbons				10.33
Monoterpenes oxygenated				86.51
Sesquiterpenes hydrocarbons				1.05
Sesquiterpenes oxygenated				0.77
Others				0.03

Tentatively identified; supported by good match according to mass spectrometry.

ND, not determined.

However, if we compare our results with those obtained in literature, we notice both qualitative and quantitative differences. Those differences consist of less borneol, α-terpineol, β-myrcene, and γ-terpinene; absence of terpinene-4-ol; detection of camphene (5.05%) for the first time; and an amount of 1,8-cineole (85.8%) that is almost double that found in M. armillaris in Australia (42.77%).⁹

Chemical differentiation of essential oil might be correlated to many factors, such as genetics, climate, soil composition, season, edaphic factors, plant organ selected, age, and vegetative cycle stage.²¹

Antioxidant activity

In light of the differences among the wide number of test systems available, the results of a single assay can give just a reductive suggestion of the antioxidant property of essential oil toward food matrices and must be interpreted with caution. Moreover, the chemical complexity of essential oil, often a mixture of compounds with scattered results, depends on the test used. For this reason, use of multiple assays in screening work is highly advisable. Different methods can be used to evaluate the antioxidant activity (ferric reducing ability of plasma, oxygen-radical absorbance capacity assay, ABTS), DPPH, and β-carotene bleaching), ensuring a better comparison of results and covering a wide range of possible applications.^22,23

Taking this into account, we evaluated the in vitro antioxidant activity of M. armillaris essential oil by using 2 methods based on free radical–scavenging capacity: DPPH radical–scavenging assay and ABTS radical–scavenging assay. Testing of this essential oil for antioxidant activity does not appear to have been reported in the literature. The essential oil's DPPH-scavenging ability (IC₅₀, 2183.6±44.3 mg/L) was less than that of ABTS (IC_50, 247.3±3.9 mg/L [Table 2]). The effect of antioxidants on DPPH radical scavenging was thought to be due to their hydrogen donating ability or radical-scavenging activity. When a solution of DPPH is mixed with that of substance that can donate a hydrogen atom, DPPH is reduced to diphenypicrylhydrazine (nonradical) with the loss of this violet color.¹¹ ABTS is used as a free radical to evaluate the antioxidant activity of a sample. The method is based on the ability of antioxidant molecules to quench the long-lived ABTS radical cation (ABTS^+•).¹² By using 2 tests, it is therefore possible to draw a firmer conclusion on the oxidative nature of the oil and its components.

Table 2.

Biological and Antioxidant Activities of Melaleuca armillaris Essential Oil

Sample	Cytotoxic activity	Antiplasmodial activity	ABTS assay	DPPH assay
Essential oil	12±1	27±2	247.3±3.9	2183.6±44.3
Vitamin C			1.9±0.1	4.4±0.2
Chloroquine	NT	0.14^a
Doxorubicin	0.22	NT

Values are the mean (±standard deviation) of 3 independent experiments, expressed as the IC₅₀ (mg/L).

No standard deviation because the values are tested every 2 months.

ABTS, 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate; DPPH, 1,1-diphenyl-2-picrylhydrazyl; NT, not tested.

It is well known that antioxidant properties are very often related on the presence of an easily oxidizable portion on the molecule.^3,24 In fact, Ruberto and Baratta²⁴ reported that 1,8-cineole had no antioxidant activity according to thiobarbituric acid reactive substances assay. According to their report, monoterpenes hydrocarbons presented antioxidant activity because of the presence of strongly activated methylene groups, namely terpinolene, α-terpinene, and γ-terpinene; to a lesser extent, sabinene, a bicyclic one, showed good activity. Therefore, activity of the essential oil could be attributed to low amounts of these components present in the essential oil. Thus, these activities may be the consequence of a synergism of all molecules, both minor and main constituents.²³

Antimalarial activity

Malaria is a worldwide problem, and the increasing spread of a P. falciparum strain that is resistant to standard treatments has prompted numerous studies aimed at identifying new antimalarial agents. Essential oils have been successfully tested as antimalarial drugs in a mouse model of malaria.²⁵ To our knowledge, essential oils from M. armillaris or all species of Melaleuca have not been tested against malaria. The in vitro inhibitory effect of M. armillaris essential oil against the chloroquine-resistant P. falciparum (FCB1) was an IC₅₀ of 27±2 mg/L. This essential oil is considered active against P. falciparum. When compared with essential oils from other origin (Cochlospermum tinctorium ²⁶ and Lippia multiflora,²⁷ for example), these values were at a good level. The effect showed by this essential oil might be attributable to its higher monoterpene content (96.84%). In addition, minor components could have contributed to the antiplasmodial activity of the oil. It is possible that the minor components may be involved in some type of synergism with the other active compounds.²⁸

Cytotoxicity against MCF7 human breast cancer cells

In recent years, considerable attention has focused on identifying naturally occurring substances capable of inhibiting, retarding, or reversing the process of multistage carcinogenesis. Plant essential oils are believed to reduce the risk for cancer when used in prevention.²⁹

Cytotoxic activity (IC_50, 12±1 mg/L) against MCF7 human breast cancer cells of M. armillaris essential oil was shown for the first time. This high cytotoxic value suggested that the monoterpenes could be responsible for this activity. To our knowledge, very few compounds found in M. armillaris have been tested for cytotoxic properties. However, it has been reported in the literature that α-cadinol (0.02% in our essential oil) showed selective toxicity against the human colon adenocarcinoma cell line HT-29,³⁰ that β-elemene (0.26% in our essential oil) was cytotoxic against tumor cells,³¹ and that β-caryophyllene (0.05% in our essential oil) was cytotoxic against A-549- Hela and HT-29 tumoral cells.³² Moreover, the weak relative concentration of those compounds cannot fully explain the important activity of M. armillaris essential oil, suggesting that other components were active.

Conclusion

This study analyzed the essential oil hydrodistilled from leaves of M. armillaris and assessed some of its biological activities. The essential oil was found to be rich in monoterpenic compounds, such as 1,8-cineole (85.8%) and camphene (5.05%). Moderate antioxidant activity was found by using 2 complementary methods: the DPPH and the ABTS assays. In contrast, we saw activity of M. armillaris essential oil against P. falciparum and, moreover, against the MCF7 breast cancer cell line.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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