Thymus Vulgaris Against Alternaria Citri E-Gnosis, Núm

e-Gnosis E-ISSN: 1665-5745 [email protected] Universidad de Guadalajara México

Soto Mendívil, Erica A.; Moreno Rodríguez, Juan F.; Estarrón Espinosa, Mirna; García Fajardo, Jorge A.; Obledo Vázquez, Eva N. Chemical composition and fungicidal activity of the essential oil of Thymus vulgaris against Alternaria citri e-Gnosis, núm. 4, 2006, p. 0 Universidad de Guadalajara Guadalajara, México

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How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative © 2006, e-Gnosis [online] Vol. 4, Art. 16 Chemical composition and fungicidal…Soto M. E. et al. CHEMICAL COMPOSITION AND FUNGICIDAL ACTIVITY OF THE ESSENTIAL OIL OF Thymus vulgaris AGAINST Alternaria citri

COMPOSICIÓN QUÍMICA Y ACTIVIDAD FUNGICIDA DEL ACEITE ESENCIAL DE THYMUS VULGARIS L. CONTRA ALTERNARIA CITRI

Erica A. Soto-Mendívil 1, Juan F. Moreno-Rodríguez 2, Mirna Estarrón-Espinosa 3, Jorge A. García-Fajardo 3 and Eva N. Obledo-Vázquez 3

[email protected] / [email protected] / [email protected] / [email protected] / [email protected]

Recibido: abril 07, 2006 / Aceptado: noviembre 15, 2006 / Publicado: noviembre 23, 2006

ABSTRACT. An essential oil extracted from thyme (Thymus vulgaris L.) was chemically analyzed by Gas Chromatography/Mass Spectroscopy and evaluated for its fungicidal activity. The main constituents were borneol (28.4%), thymol (16.6%), carvacrol methyl ether (9.6%), camphene (6.9%), α-humulene (6.4%) and carvacrol (5.0%). Tests of fungicidal activity (in vitro) indicated that a concentration of 1000 ppm of thyme´s essential oil was effective to inhibit Alternaria citri, when added to a potato dextrose agar culture medium.

KEYWORDS. Thymus vulgaris, Lamiaceae, Borneol, Essential oils composition, Alternaria citri.

RESUMEN. Se analizó químicamente el aceite esencial de tomillo (Thymus vulgaris L.) por Cromatografía de Gases/Espectroscopia de Masas y se evaluó su actividad fungicida. Los principales constituyentes fueron borneol (28.4%), timol (16.6%), carvacrol metil eter (9.6%), camfeno (6.9%), α-humulene (6.4%) y carvacrol (5.0%). Las pruebas de actividad fungicida (in vitro) indicaron que la concentración de 1000ppm del aceite esencial de tomillo fue efectivo para inhibir a Alternaria citri, cuando se adicionó al medio de cultivo agar papa dextrosa.

PALABRAS CLAVE. Thymus vulgaris, Lamiaceae, Borneol, Composición de aceites esenciales, Alternaria citri.

Introduction

Essential oils are chemical products formed by odoriferous essences extracted from a great diversity of plants. They are volatile liquid, insoluble in water, highly soluble in alcohol, ether, vegetable and minerals oils. Essential oils are complex mixtures of different chemical compounds. However, they can be characterized by their main volatile components [1, 2]. The proportion of the volatile components varies from one essential oil to another, even within species, providing to each species different toxic and medicinal properties. The proportion of the volatile components varies in the plants depending upon season, being generally higher during the summer [3].

It is not completely known the exact function of essential oils in plants; some functions attributed are: attraction of insects for pollination, insect repellent, or intermediary metabolic products. Initially, essential oils were considered as waste material of plant metabolism; however, currently their biological importance

1 Universidad de Guadalajara, Centro Universitario de Ciencias Exactas e Ingenierías. Posgrado en Procesos Biotecnológicos. www.cucei.udg.mx/˜posbio/ 2 Universidad de Guadalajara, Centro Universitario de Ciencias Exactas e Ingenierías. Departamento de Química 3 Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A. C. www.ciatej.net.mx

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© 2006, e-Gnosis [online] Vol. 4, Art. 16 Chemical composition and fungicidal…Soto M. E. et al. has been recognized [4-6]. In the past, essential oils were only used for therapy purpose directly for humans and animals. More recently the antimicrobial properties of essential oils are also utilized in food industry, to prolong shelf life of food products [7-9]. Essential oils also are used to add aroma and flavor for coffee, tea, wines and alcoholic drinks. They are basic ingredients in perfume's industry to prepare soaps, disinfectant, antioxidant and similar products [10-11]. Essential oils are important in medicine for their pleasant flavor, palliative effect to the pain and their low toxicity [12].

Thyme (Thymus vulgaris L.) belongs to the Lamiaceae family, and is an aromatic and medicinal plant very important for the horticultural industry. Thyme is a perennial shrub with woody stems and it reaches a height of 30-50 cm. It is a typical plant of the Mediterranean region. Thyme possesses antiseptic properties, antispasmodic, carminative, diaphoretic, disinfectant, deodorant, diuretic, expectorant, sedative, tonic and antihelmintic. Its properties are due to its main components: thymol and carvacrol [13]. However, there are several chemotypes for thyme, such as: geraniol, linalool, α-terpineol, thymol, carvacrol, p-cymene, trans- sabinene hydrate, terpinene-4-ol, borneol and 1, 8-cineole, most of them with antimicrobial activity [3].

In recent years, several studies have been carried out with this plant to test its antimicrobial activities [5, 9, 14-17]. Specifically, the study of essential oil of Thymus capitatus has shown strong fungicidal activity against Alternaria citri, affecting elongation of the germinative tube [18].

Mexico is the principal mexican lime (Citrus aurantifolia, Swingle) producing country. During the last twenty years, state of Michoacán has been the most increase in production, 12,514 ha in 1980 to 29,733 ha in 2000. The principal phytosanitary problems in this crop are infectious (biotic) and non-infectious (abiotic) diseases [19, 20]. Different diseases exist in this crop due to the great diversity of agroecological characteristics exists in Mexico. Alternaria citri is a serious problem in citrus production world wide. Currently, this is an important phytopathogen in the state of Michoacán causing the Alternaria black rot of citrus plants, a disease that can significantly reduce the yield, and it can be a serious problem for the fresh fruit market as well as for the processing industry, since a small portion of rot in a fruit imparts a bitter flavor [21]. Benomyl (Benlate) and thiabendazole (TBZ) are synthetic benzimidazole fungicides effective against this disease; however, Benlate has been discontinued [22]. Thymus vulgaris has already been reported among the natural sources of antimicrobial compounds, [14, 15]. The objective of this work was to obtain and characterize the essential oil of Thymus vulgaris and to evaluate in vitro its fungicidal activity against Alternaria citri isolated from mexican lime cultivated in the state of Michoacán.

Materials and methods

Plant material and distillation: Dried leaves of thyme (Thymus vulgaris L.) were acquired at a local market in Guadalajara, Jalisco, Mexico. The extraction was carried out using a water steam stainless steel distillation equipment (60 L) with recirculation of water. Leaves were distilled during 2 hours and the extracted essential oil was dried over anhydrous sodium sulfate. Then, the oil was stored in sealed amber flasks at 5°C.

Physicochemical analysis: The content of the essential oil (V03-409), moisture content (V03-402) and total ashes (V03-403) were determined in the leaves according to French AFNOR norms [23]. Solubility in ethanol (T75-101), relative density at 20°C (T75-111), and the refraction index (T75-112) [24] were determined for the collected essential oil.

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Determination of composition of essential oil: Gas chromatography was used for the qualitative and quantitative determination of oil composition. Qualitative analysis was performed by Gas Chromatography/Mass Spectroscopy (GC/MS) using a GC HP 5890 series II coupled with mass selective detector (HP 5972). Separation was carried out in a capillary column DB-Innowax (J&W Scientific) (60m x 250µm ID x 0.25µm film thickness). Injector and detector temperatures were 250°C and 280°C, respectively. Oven temperature was 60°C, then raised to 240°C at 3°C/min and held for 20 min; the carrier gas was helium at a flow rate of 0.7 mL/min. Injection volume was 0.5 mL using a split ratio of 1:200. The mass spectrometer was operated in mode SCAN with an ionization voltage of 70 eV scanning from m/z 30- 400 at 0.7 scans/s. Volatile components were identified by comparison with mass spectra library Wiley 275L (Rev. C.00.00) and when it was possible, with commercial standards (Sigma-Aldrich 95% min). The elution order was verified by Kovats Index in similar phases, as reported in the literature [25, 26].

Quantitative determination of components of the essential oil was performed using a GC HP 6890 series GC system with flame ionization detector (GC/FID). Separations were carried out in a capillary column DB- Innowax (J&W Scientific) (60m x 250µm ID x 0.25µm film thickness). Injector and detector temperatures were 250°C and 260°C, respectively. The oven temperature program was from 60°C to 240°C at a rate of 3°C /min, then the temperature was held for 15 min. Helium was the carrier gas with a flow of 0.9 mL/min. The injected volume was 0.5mL with a split ratio of 1:200. Components were quantified as area percentage of total volatiles from electronic integration data (HP GC Chem Station A.06.03).

Determination of fungicidal effect of the essential oil

Biological material: The strain of Alternaria citri was obtained from the Agrobiology Faculty of the University: Michoacana de San Nicolás de Hidalgo in Uruapan, Michoacán, Mexico.

Bioassays: Five concentrations of thyme essential oil (10, 50, 100, 1000 y 10000 ppm) were prepared in Petri plates with potato-dextrose-agar (PDA-Bioxon®). The inoculation consisted of cutting a circle of culture medium with fungal growth (Alternaria citri) by using a test tube of 1 cm diameter, and then the circle was transferred to a new Petri plate with PDA by means of a nicromo handle. Subsequently, plates were incubated at 23 ± 3°C with their respective controls, until the fungus completely covered the control plate (15 days). Radial colony growth was measured with a graduated rule. The experimental design used was unifactorial with five treatments (essential oil concentration levels). The experiment was carried out twice, (n= 8).

Results and discussion

Analysis of Thymus vulgaris leaves. The content of total essential oil in Thyme leaves was 2.05% (Table 1). In a production trial, on a pilot scale, the yield of essential oil was 1.7% on dry weight basis. Therefore, oil recovery from this process was 83%.

Table 1. Chemical characteristics of Thymus vulgaris leaves. Characteristic Raw material AFNOR** Essentials oils content * 2.05 % ≥1 Moisture content 8.75% ≤12 Total ashes * 6.66% ≤14 • Expressed in dry weight. **French AFNOR norm [23]

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The content of total essential oil in thyme leaves was 2.05% (Table 1). In a production trial, on a pilot scale, the yield of essential oil was 1.7% on dry weight basis. Therefore the oil recovery from this process was 83%.

The physical characteristics of thyme essential oil are presented in Table 2.

Table 2. Physical characteristics of thyme oil (Thymus vulgaris) Characteristic Value AFNOR** Solubility in ethanol 1 vol. at 85% ≥2V al 80% Relative density at 20°C 0.939 0.910-0.935 Refraction index at 20°C 1.4914 1.495-1.505 **French AFNOR norm [24]

Characteristics of the current sample closely conform the French standard [23, 24]. It is not uncommon to find variations in physical characteristics of essential oil. The solubility of the essential oil of Thymus vulgaris was reported as 1 to 1.5 vol (or higher) at 80% alcohol [27], which is lower than the present standard. Similar value was obtained (1 vol at 85%) in this study. The refraction index obtained in this study is slightly lower than the french standard. These type of variations are reportedly common and are due to the variation in chemotypes, stage of plant life cycle, different parts of the plant, as well as environmental factors as season, cultivation, and storage conditions [28].

The chemical composition of the essential oil obtained was analyzed by GC-FID and GC/MS, which allowed identification of 27 volatile compounds (Table 3). The main compounds were borneol (28.4%), thymol (16.6%), carvacrol methyl ether (9.6%), camphene (6.9%), α-humulene (6.4%), and carvacrol (5.0%).

Table 3. Volatile composition of thyme essential oil (Thymus vulgaris) # Peak Compound KI % Area Identification 1 Tricyclene 1009 0.3 KIa, MS 2 α-Pinene 1039 4.2 KIab, MS, STD 3 Camphene 1083 6.9 KIab, MS, STD 4 β−Pinene 1124 0.7 KIab, MS, STD 5 Myrcene 1156 0.4 KIab, MS, STD 6 α−Terpinene 1189 0.4 KIa, MS, STD 7 Limonene 1206 0.5 KIab, MS, STD 8 1,8-Cineole 1228 0.1 KIab, MS, STD 9 γ-Terpinene 1251 1.7 KIab, MS, STD 10 p-Cymene 1272 2.4 KIab, MS, STD 11 α-Terpinolene 1287 0.1 KIab, MS, STD 12 cis-Sabinene hydrate 0.2 MS 13 α-Copaene 1493 0.3 KIa, MS 14 Linalool 1506 0.5 KIab, MS, STD 15 Camphor 1518 3.0 KIab, MS 16 Isobornyl acetate 1584 0.3 KIab, MS 17 Bornyl acetate 1599 1.4 KIa, MS, STD 18 Carvacrol methyl ether 9.6 MS

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19 α-Humulene 1682 6.4 KIb, MS, STD 20 Borneol 1698 28.4 KIab, MS, STD 21 α-Muurolene 1730 0.1 KIab, MS 22 δ-Cadinene 1761 0.6 KIab, MS 23 γ-Cadinene 1785 0.4 KIab, MS 24 cis-Calamenene 1837 0.0 KIa, MS 25 Caryophyllene oxide 1966 0.7 KIa, MS, STD 26 Thymol 2100 16.6 KIb, MS, STD 27 Carvacrol 2159 5.0 KIab, MS, STD Total 91.2 KI, Kovats index a[25], b[26]; MS, comparison of mass spectra); STD, co-injection with authentic compounds.

Several past studies identified thymol, carvacrol and p-cymene as the main components in Thymus essential oil [3, 17, 29, and 30]. However the composition of essential oil is affected by the factors mentioned earlier and great chemical intraespecific variability among plants is also known in this specie [31]. Furthermore an important variation of essential oil composition was reported during the vegetative cycle of Thymus vulgaris [28]. For instance, Borneol was noted to show more than 200% variation, carvacrol 180%, thymol 50%, and p-cymene variations 177% at different stages of life cycle of this plant [28]. In this study, the sample used demonstrated high level of borneol, and therefore, it is suggested that Thymus vulgaris in this case is a borneol chemotype.

The in vitro tests showed that the essential oil of thyme was effective against mycelial growth of Alternaria citri. At a concentration of 1000 ppm or higher, this essential oil was able to inhibit growth of the fungus. There were significant differences (α<0.05) among the concentrations tested (Fig. 1). ) m c (

4 h t w o

r 3 g

l a i 2 d a r

y 1 n o l o

C 0 Control 10 50 100 1000 10000 ppm

Figure 1. Effect of thyme essential oil (Thymus vulgaris) on the radial growth of Alternaria citri on PDA.

Even though the essential oil of thyme has been recognized by its antimicrobial activity attributed mainly to thymol and carvacrol, this essential oil (chemotype borneol), has demonstrated fungicidal activity against Alternaria citri in culture medium. The antifungal activity of this essential oil could be due to thymol and carvacrol as well as borneol or a synergistic effect between some of their components. The individual component of these three also demonstrated antimicrobial activity [17, 32]. Thymus vulgaris essential oil when contained 2-11% of carvacrol and 10-64% thymol showed antimicrobial activities [17]. The essential

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© 2006, e-Gnosis [online] Vol. 4, Art. 16 Chemical composition and fungicidal…Soto M. E. et al. oil extracted in this study contained carvacrol and thymol in the range reported above. Other plants including Micromeria cristata and Thymus pectinatus have been shown to have antimicrobial activities when they contain borneol as the main component, but missing thymol and carvacrol [32, 33]. Synergetic antimicrobial effect had been reported among carvacrol and its precursor p-cymene [17].

Conclusion

Characterization of essential oil of Thymus vulgaris was carried out, and its antifungal activity against Alternaria citri was demonstrated. This antifungal activity may be attributed to the essential oil compounds borneol, thymol, and carvacrol. Thymus vulgaris essential oil shows potential to be utilized in developing organic fungicides against Alternaria black rot in Citrus.

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