MINUTA L. AND AREIRA L. ESSENTIAL OILS AS

ALLELOPATHIC AGENTS.

Lidia R. Scrivanti1, María P. Zunino2 and Julio A. Zygadlo1

ABSTRACT. The bioassay with headspace of T. minuta and S. areira oils and their pure principal components revealed strong inhibitory activity of the root growth of Zea mays seedling. Both T. minuta and S. areira oils treatment presented an increase in malondialdehyde values from 24 to 48 h, while the main components of the essential oils, ocimenone, α pinene and limonene, presented an increase from 24 to 96 h indicating exhuasted lipid peroxidation.

The T. minuta had a strong inhibitory action and oxidant effect on root of Zea mays than S. areira oil.

Key Word Index – L.; ; Schinus areira L.; ; allelopathy; headspace terpenes.

INTRODUCTION

The occurrence of deleterious biochemical interactions among higher

(allelopathy) is generally accepted as a significant ecological factor in determining the structure and composition of communities. Reports of allelopathic relationships are numerous

(Lorber and Muller, 1976; Fischer, 1986; Einhellig and Leather, 1988; Mizutani, 1989; Seigler,

1996). The most frequently found allelochemicals are phenolic compounds and terpenoids

______1Instituto Multidisciplinario de Biologia Vegetal, Catedra de Química Orgánica, Facultad de Ciencias Exactas, Físicas y Naturales. Avenida Velez Sarsfield 1600, 5000 Córdoba, . [email protected].; [email protected]. tel/fax 054-0351-4334439. 2Fellowship from Consejo Nacional de Investigaciones Cientificas y Tecnicas. [email protected]

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( Einhellig and Leather,1988; Wójcik-Wojtkowiak, 1992). Plant volatile oils have been recognized since antiquity to possess biological activities.

Chief amongst these are their antibacterial, antifungal and antioxidant properties (Lorber and

Muller, 1976; Demetzos et al., 1995; Dorman et al., 1995; Zygadlo et al., 1995; Bishop andThornton, 1997).

Some of the essential oils considered to have greatest bioactivities are those of the following plants: Tagetes minuta L. and Schinus areira L. (Héthélyi et al., 1986; Fischer, 1991; Zygadlo et al., 1994).

Different physiological and biochemical changes were studied by allelopathic action of essential oils (Asplund, 1968, Lorber and Muller, 1976, El-Deek and Dan Hess, 1986; Fischer,

1986, Fischer, 1991; Koitabashi et al., 1997). Lipid peroxidation, widely recognized as a primary toxicological event, is caused by the generation of free radicals from a variety of sources including organic hydroperoxides and redox processes. The secondary events, include changes in membrane structure, permeability and fluidity, lysosomal destabilization and stimulation of apoptosis (Dorman et al., 1995, Sikkema et al., 1995). Thus, we propose a correlation between lipid peroxidation and growth inhibition of roots.

The monoterpenes have negligible water solubility compared to other classes of organic compounds. Several studies of allelopathy have proposed that natural detergents may speed solubilization and increase the solubilities of terpenoid compounds (Weidenhamer et al., 1993).

However, the monoterpenes are thought to be important allelopathic agents in hot, dry climates where they act in the vapour phase, because the high vapour density of the essential oils may penetrate into soil, affecting adversely the undergrowing plants (Kohli and Singh, 1991; Vaughn and Spencer, 1993; Koitabashi, et al. 1997).

In this study, the essential oils and their principal components in the vapour phase from the two plants started above were evaluated for their oxidant and allelopathic properties on root growth.

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EXPERIMENTAL.

Plant material. Tagetes minuta L and Schinus areira L were collected at the flowering stage in

Córdoba, province of Argentina. Vouchers (Scrivanti 10 and 11, respectively, CORD) are on deposit at the herbarium of Museo Botánico de Córdoba.

Essential oils and terpenes. Essential oils of Tagetes minuta L. and Schinus areira L. were hydrodistilled as described by Zygadlo et al. (1993). Limonene and α-Pinene were purchased from Aldrich. Ocimenone was isolated from Tagetes minuta essential oil by TLC (Stahl, 1969).

Plant material. Maize seeds (Zea mays L.) were rolled in the upper 3 cm of 20 cm tall paper towel scrolls. The individuals scrolls were moistened with 25 ml of dist. H2O and placed upright in 3 l flasks. The scrolled seeds were germinated for 3 days at 27 ± 10C in the dark. At the end of this period the length of the roots was 6 ± 1 cm and the seedlings were harvested and then were transferred to bioassay.

Bioassay. The 3 day seedlings were place in 3 l desiccator flasks and in the central area a glass beaker of 5 ml was placed. A sample of 2 ml of T. minuta or S. areira essential oils (volatile source) was added to the beaker and in the case of principal components we worked with the minimum inhibitory concentration. The minimum inhibitory concentration was defined as the concentration which inhibit the 50% of roots growth at 96 h of treatment compared with the control. These were 29 mg/l, 27 mg/l and 18 mg/l for limonene, α-pinene and ocimenone, respectively. The flasks were placed in darkness at 27 ± 1°C for 24, 48 and 96 h. After which times plants were harvested and the roots were dissected and measured. The central beakers were left empty in the controls.

Volatile analysis. Volatiles from headspace of glass desiccators were trapped by using a 10 ml gastight syringe and were analyzed by GC and GC/MS. A GC (Shimadzu R1A) equipped with a flame ionization detector was used to analyze volatiles. A split inlet (split ratio 200:1) was used to inject volatiles into a DB-5 capillary GC column (0.25 mm id x 30 m, and 0.25 μm film thickness), and ramped column temp. conditions (600C for 3 min, increased to 2400C at

3 40C/min) were used. Detector temp. was 2800C. He was the carrier gas with a constant flow of

0.9 ml/min. Individual peaks were also identified using a mass selective detector (GC/MS

Perkin Elmer Q700) and co-injection with standards. GC/MS was performed with same conditions as GC. The ionization potential of MS was 70 eV. The quantity of essential oil and their principal components in the headspace was determined by the external standard method.

The standard curves were generated by analysis of known concentrations of each compound dissolved in n-C6H14.

Thiobarbituric acid assay. The advantages and limitations of this method were discussed by

Schmedes and Holmer (1989). Roots (100 mg) were homogenized in 5 ml of EtOH (96% v/v).

An equal volume of 0.5% 2-thiobarbituric acid in 10% trichloroacetic acid solution was added and the sample incubated at 950C for 30 min. The reaction was stopped by putting the reaction tubes in an ice bucket. The samples were then centrifuged at 10000 g for 30 min. The supernatant was removed and read at 532 nm (Schmedes, et al. 1989, Cheftel et al., 1992). The amount of malondialdehyde present was calculated from the extinction coefficient of 155 mM

(Kosugi et al.,1989).

Estimation of peroxides. This method was used to measure the oxidation state of the principal components from the essential oils studied.

One hundred μl of each terpenes were transferred to a 16 x 150 mm test-tube. Twenty-five μL of amonium thiocynate solution and 4.85 ml of chloroform: methanol (3:5) are then added.

After 10 min the tube was read at 505 nm. The peroxide value is then calculated as milliequivalents per kg of terpene using the extinction coefficient of 55.84 (Chapman and

Mackay, 1949).

Data analysis. Data were analyzed using ANOVA with INFOSTAT pragramme.

Whenever ANOVA indicated significant effects (p<0.05), a pairwise comparison of means by

Duncan’s multiple range test was carried out.

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RESULTS AND DISCUSSION.

The composition of the headspace for T. minuta and S. areira essential oils are shown in

Table 1. Thus, α-pinene (85.3%) and limonene (66.3%) with ocimenone (21.8%) were the major constituents of S. areira and T. minuta headspace oils, respectively.

Although no single structural feature of monoterpenes has appeared to be a critical factor in inhibiting germination, several of the most phytotoxic compounds contained a chemical group with oxigen (such as carbonyl, carboxil or alcohols) while several of the least toxic were hydrocarbons (Vaughn et al. 1993; Asplund, 1968). However, interactions with cyclic hydrocarbon monoterpenes lead to changes in structure and function of the membranes, which in turn, may impair growth and activity of the cells (Sikkema et al., 1995). Thus, the bioassays with T. minuta and S. areira oils revealed strong inhibitory activity of the root growth not showing development of the root from 24 to 96 h of treatment. Moreover, among the oil tested, that of T. minuta oil was most effective phytotoxic than S. areira oil, because T. minuta oil inhibited more, the growth of roots, than S. areira oil (Table 2).

In previous studies, plants exposed to monoterpene vapours have shown severe internal damage. The absence of a variety of intact organelles and the presence of membrane fragments indicate that structural breakdown and descomposition are occurring within inhibited root

(Lorber et al.,1976). As fatty acids and other lipids are known as structural components of membranes, it is reasonable to suppose that membrane degradation could result in the freeing of lipids within the cytoplasm of targeted cells. Then, the free lipids within the cytoplasm could be the target of an oxidative action. The effect of the essential oils as “oxidant” agent is shown in

Table 3. As can be seen, the treatments presented an increase in malondialdehyde values from

24 to 48h. This suggests that lipid peroxidation is occurring. After 48 h the level of malondialdehyde declined, there are two likely reason a) it is oxidized further, b) the precursor of unsaturated fatty acid is decreased (Stewart and Bewley, 1980).

5 There is an increased lipid peroxidation during senescence of plant tissues (Dhindsa et al., 1981). For these reasons, the correlation coefficient between malondialdehyde concentration and root length of the control seedlings was strong and positive (r = 0.99). The correlation coefficient of seedlings treated with T. minuta oil was seen to be strong and negative (r =- 0.90), however we did not find a strong correlation with S. areira oil (r = 0.43). From results of correlation it can be proposed that the phytotoxic action of T. minuta is primarily the result of increased lipid peroxidation rates. While the growth inhibition in S. areira may be accounted for in other ways, as for example the cineole that inhibits DNA synthesis (Koitabashi et al., 1997).

In Tables 4 and 5 are shown the effect on the roots growth and MDA values respectively, of the main components of each other essentisl oils studied (α-pinene, limonene and ocimenone). Limonene and ocimenone inhibited the roots growth from 24 to 96 h, while the growth with α-pinene was inhibited only in the first 24 h of treatment.

The MDA values were high for ocimenone and limonene since 24 h of treatment, while with α-pinene the MDA values were hyghly significative from 48 h.

The essential oils suffer oxidation processes too (Hausen et al, 1999; Pokorny et al,

1998), and it is possible that these products may have phytotoxic activity. There were not found hydroperoxides in the essential oils and their main components studied (data not shown), suggesting in this way that the essential oils did not oxidized during the period and in the conditions that this experence was carried out. In this way, the effects on the growth inhibition of roots and oxidation values, are due to the essential oils and their main components, and not to their autooxidation products. Even though this products could be formed subsequently inside the cell.

Finally, we can conclude that from the terpenes studied the ocimenone was the compound that presented the most inhibitory action on the root growth and the highest oxidation values. Is so that T. minuta essential oil is more phytotoxic, for its high content in ocimenone, that S. areira essential oil.

6 Acknowledgement. AGENCIA CBA CS., CONICET and SECYT-UNC.

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Table 1. Composition (in %) of the headspace of Tagetes minuta and Schinus areira oils. Compounds T. minuta S. areira

Camphene 10.8 Carvone 0.1 Limonene 66.3 2.8 α-Pinene 11.8 85.3 β-Pinene 1.7 (E) Ocimenone 19.1 (Z) Ocimenone 2.7

Total essential oil (μg/l) 20.8 41.3

Table 2. Effect of the two essential oils on Zea mays root growth. Root length (cm) Treatment

Time (h) Control Tagetes minuta Schinus areira

10 24 9.77 ± 0.4 a1 5.86 ± 0.2 a2 5.48 ± 0.1 a3 48 11.76 ± 0.5 b1 5.72 ± 0.1 a2 6.19 ± 0.2 b3 96 15.13 ± 0.5 c1 6.18 ± 0.3 b 2 6.09 ± 0.2 b2 Values (means ± SD) having different letter in the same column and different numbers in the same row are significantly different from each other according to Duncan’s multiple range test at p: 0.05 (n=20).

Table 3. Effect of the two essential oils on malondialdehyde concentration of the Zea mays root growth during the germination period. Malondialdehyde concentration (µmol / g dry mass)

Treatment

Time (h) Control Tagetes minuta Schinus areira 24 0.88 ± 0.05 a1 1.04 ± 0.06 a2 1.03 ± 0.02 a2 48 1.20 ± 0.08 b1 1.75 ± 0.12 b2 1.36 ± 0.04 b3 96 1.36 ± 0.06 c1 0.75 ± 0.06 c2 0.94 ± 0.04 c3

Values (means ± SD) having different letter in the same column and different numbers in the same row are significantly different from each other according to Duncan’s multiple range test at p:0.05 (n=5).

Table 4. Effect of the three monoterpens on Zea mays root growth. Root length (cm) Treatment

Time (h) Control Ocimenone Limonene Pinene

24 9.77 ± 0.4 a1 6.17 ± 0.4 a2 7.2 ± 0.5 a3 8.21 ± 0.3 a4 48 11.76 ± 0.5 b1 7.12 ± 0.8 a2 10.6 ± 0.5 b3 11.07 ± 0.5 b1 96 15.13 ± 0.5 c1 8.53 ± 0.9 b 2 13.56 ± 0.2 c3 14.01 ± 0.2 c1 Values (means ± SD) having different letter in the same column and different numbers in the same row are significantly different from each other according to Duncan’s multiple range test at p: 0.05 (n=20).

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Table 5. Effect of the three monoterpens on malondialdehyde concentration of the Zea mays root growth during the germination period. Malondialdehyde concentration (µmol / g dry mass)

Treatment

Time (h) Control Ocimenone Limonene Pinene

24 0.88 ± 0.05 a1 1.35 ± 0.09 a2 0.98 ± 0.05 a3 0.87 ± 0.04 a1 48 1.20 ± 0.08 b1 1.66 ± 0.05 b2 1.42 ± 0.09 b3 1.32 ± 0.02 b3 96 1.36 ± 0.06 c1 2.51 ± 0.09 c2 2.20 ± 0.04 c3 1.90 ± 0.01 c4

Values (means ± SD) having different letter in the same column and different numbers in the same row are significantly different from each other according to Duncan’s multiple range test at p:0.05 (n=5).

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