Abstract
Aromatic plants are broadly used in popular medicine. Essential oils are receiving increasing attention in the pharmaceutical industry as they contain antibacterial, antifungal, anti-inflammatory, and anticancer substances. The brine shrimp (Artemia salina.) assay is a convenient preliminary toxicity test, because brine shrimp is highly sensitive to a variety of chemical substances. The essential oils of 12 vegetable species were obtained by a hydrodistillation process. Essential oils of all plants were screened for their toxicity in the brine shrimp assay with larvae of Artemia salina.. The 50% lethal concentration (LC50) values of essential oils were determined by Finney's Probit analysis. Nontoxic essential oils for Artemia salina. were Aloysia polystachia. (LC50 6459 µg/mL), Minthostachys verticillata. (Griseb.) Epl (LC50 1848 µg/mL), Aloysia triphylla. (L'Herit.) Briton (LC50 1279 µg/mL), and Schinus poligamus. (Cav.) Cabrera (LC50 1179 µg/mL). The most toxic essential oils were Psila spartoides. (H. et A) J.Rimy (LC50 14 µg/mL) and Hyptis mutabilis. (Rich.) Briq (LC50 30 µg/mL). Previous studies with the essential oils of these vegetables have shown antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, and yeast.
Introduction
Aromatic plants, an important component of Argentine flora, are widely distributed in different regions of the country and are broadly used in popular medicine by local people (Zygadlo & Juliani, Citation2001). These plants are able to produce a great variety of natural products and extracts, including essential oils. Volatile oils are very complex mixtures of aromatic compounds, primarily monoterpenes and sesquiterpenes (Svoboda & Hampson, Citation1999). These oils and their derivatives have been used for a wide variety of purposes for thousands of years, varying from perfumery, flavoring and preservation of stored food crops, to medicinal products (Bagci & Digrak, Citation1996; Hammer et al., Citation1999). It has recently been found that components of essential oils have a considerable range of applications, including antibacterial, antifungal, anti-inflammatory, and anticancer activities, and they are receiving increasing consideration by the pharmaceutical industry (Hammer et al., Citation1999; Zygadlo & Juliani, Citation2000; Demo et al., Citation2001; Demo et al., Citation2002; Burt, Citation2004). Recent attention has been given to extracts and biologically active compounds isolated from plant species used in herbal medicine in order to find a solution to the side effects of drugs and to the resistance that pathogenic microorganisms build against antibiotics.
Plant-based antimicrobials represent a vast untapped source of medicines, and further exploration of plant antimicrobials is necessary (Kokoska et al., Citation2002). Hundreds of natural substances are being isolated and identified every year, but data concerning their biological activities have been little investigated (Burt, Citation2004). To use a substance for medicinal purposes, it is necessary to meet some requirements, including its toxicity to eukaryotic cells. The brine shrimp (Artemia salina.) assay is a convenient preliminary toxicity test, because brine shrimp are highly sensitive to a variety of chemical substances. Artemia salina. is a crustacean found in saline water worldwide. The availability of eggs, the ease of hatching them into larvae, the rapid growth of nauplii, and the relative ease of maintaining a population under laboratory conditions make the brine shrimp a simple and effective test animal in bioassays and toxicology studies. All stages in its life cycle have been exposed to several chemical substances and used as criteria for toxicity. The relevance of these values is mainly due to the fact that they can be indicators of possible antitumor activities (Svoboda & Hampson, Citation1999). Toxicity to brine shrimp coincides with toxicity to mammalian cells in many cases. However, there is no correlation in the degree of toxicity between the two systems. For some compounds, toxicity to the brine shrimp test is often used as a tool for approaching the real toxicity (Franssen et al., Citation1997).
This study was focused on the toxic activity of essential oils from Argentina on Artemia salina. in order to have a preliminary value of the toxicity to eukaryotic cells.
Materials and Methods
Plant material
The aerial parts of the medicinal plants used were collected from different regions of Argentina. Specimens are kept in the herbarium of the Museo Botánico de la Facultad de Ciencias Exactas, Fisicas y Naturales of the Universidad Nacional of Cordoba (Argentina).
Essential oils obtained
The essential oils (EOs) were obtained from the vegetable material, which was hydrodistilled in a Clevenger-like apparatus. The oil obtained was kept in a partition ampoule and stored in a freezer (− 80°C) until analysis.
Cytotoxic activity of essential oils
Essential oils of all the plants sampled were screened for toxicity with larvae of Artemia salina. as described by Franssen et al. (Citation1997). Two-fold dilutions were made for the essential oils in dimethylsulfoxide (DMSO: final concentration 1/200). Artemia. eggs were incubated in a Petri dish divided by a piece of acrylic that allows larvae to swing from one side to another. The solution used was seawater (3.3% salt marine solution), and eggs were left at room temperature in darkness for 24–48 h. The nauplii were collected and brought into contact with the essential oils dilution in microwell plates. After 24 h of incubation at room temperature, the number of dead nauplii in each well was determined with a microscope. The plates were left for 12 h at − 20°C, and the number of total larvae was determined. The test was performed in duplicate. The mortality percentage was determined in relation to the number of larvae killed by the oils and the number of total larvae. Finney's Probit analysis was used to determine the 50% lethal concentration (LC50) of essential oils. Essential oils dilutions that did not show toxicity at 1000 µg/mL were considered nontoxic (Franssen et al., Citation1997).
Results and Discussion
The vegetable material collected was obtained from different regions of Argentina. Different quantities of essential oils were obtained from these materials. lists the plants collected, the place of collection, and their common names and popular uses. Twelve vegetable species were collected from three geographic regions: northern region (Provincia de Salta and Provincia de La Rioja province); central region (Provincia de Córdoba); and southern region (Provincia de Chubut).
Table 1.. Aromatic plants from Argentina
Toxicity to brine shrimp larvae was determined as previously described. The results are shown in . Toxicity of essential oils to A. salina. is poorly described, but there are reports involving ethanol extracts of vegetables that mention values of LC50 = 1000 µg/mL for them (Rojas de Arias et al., Citation1995; Franssen et al., Citation1997). For this reason, in this paper we will consider these values to determine the toxicity of the oils to the larvae.
Table 2.. The 50% lethal concentration (LC50) of essential oils and maximum tolerated concentration (MTC) on A. salina.
Aloysia polystachia. (LC50 6459 µg/mL), Minthostachys verticillata. (LC50 1848 µg/mL),Aloysia triphylla. (LC50 1279 µg/mL), and Schinus poligamus. (LC50 1179 µg/mL) showed a decreasing activity in the brine assay and are considered nontoxic. The most toxic essential oils were Psila spartoides. (LC50 14 µg/mL) and Hyptis mutabilis. (LC50 30 µg/mL). It is also important to note that Lantana xenica. (LC50 995 µg/mL), Ophryosporus charrrua. (LC50 972 µg/mL), and Anemia tomentosa. (LC50 968 µg/mL) toxic values were very near the nontoxicity limit value, taking into consideration that the oils of the three vegetables showed antimicrobial activity in previous studies (Juliani et al., Citation2002; Demo et al., Citation2005).
In our previous studies, A. polystachia., A. triphylla, M. verticillata., and S. poligamus. showed antimicrobial activities against Gram-positive and Gram-negative bacteria. A. polystachia. was active against Staphylococcus aureus, Bacillus cereus., and Proteus mirabilis. (Primo et al., Citation2001; Gonzalez et al., Citation2004; Demo et al., Citation2005). It is important to note that A. polystachya. was nontoxic at the maximum tolerated concentration (MTC; 3530 µg/mL). The essential oils of A. triphylla. were active against Gram-positive bacteria, such as S. aureus, S. epidermidis, B. cereus, Micrococcus luteus, Enterococcus faecalis., and Gram-negative bacteria, such as Escherichia coli, Klebsiella pneumoniae., and P. mirabilis. (Demo et al., Citation2005). S. aureus. is responsible for human diseases and is a food-contaminating microorganism, as is B. cereus., a spore-forming bacilli. It is also important to note that in previous studies A. triphylla. showed a remarkable antifungal activity against Candida albicans. (Hammer et al., Citation1998; Demo et al., Citation2005). As a consequence, this nontoxic essential oil could be considered as a potential antifungal agent against mucous and cutaneous candidiasis. The main components of the essential oil of A. triphylla. identified were limonene, neral, geranial, spathulenol, and thujone (Oliva, Citation2005). Thujone is considered a toxic compound with symptoms associated with epileptiform convulsions with general vasodilatation, hypotension, lower cardiac rhythm, and increased respiratory amplitude. The European Union sets the maximum levels for thujone (α and β) in foodstuffs and beverages at 0.5 mg/kg, whereas the United States does not authorize its use as a flavoring substance (Scientific Commitee on Food, Citation2003). Considering the lack of toxicity obtained in this study with A. triphylla. oil, it could be suggested that the components included in the essential oil interact chemically among themselves and neutralize the toxic effects of thujone in the oil.
This essential oil of M. verticillata. was active against Gram-positive and Gram-negative bacteria. The main components identified in the essential oils where pulegone, menthone, limonene, and α- and β-pinene (Primo et al., Citation2001). Pulegone is considered hepatotoxic (The European Agency for the Evaluation of Medicinal Products, Citation2004), but a study revealed that toxicity of pulegone was suppressed in the presence of menthone, indicating antagonistic interactions (Pavlidou et al., Citation2004). The whole oil of M. verticillata. was nontoxic to A. salina. (DL50 1848 µg/mL), consequently it should be taken into consideration for antimicrobial therapeutic formulations.
The ease of growing and the facility of use in the laboratory makes A. salina. an attractive organism to work with. The toxicity test with this larvae could be considered useful and reliable to check the toxicity of other substances with different biological effects. Toxicity to Artemia. could be similar to the toxicity to mammalian cells. It is a useful test for approaching nontoxic concentrations to work with mammalian systems (Logrieco et al., Citation1996; Franssen et al., Citation1997).
During the past few years, there has been an important increase in the consumption of aromatic plants. Because very few species are cultivated, they are mainly collected from the wild to meet the growing demands of industry. As a result, natural populations are declining and genetic diversity is being lost (Zygadlo & Juliani, Citation2001). The results in this paper clearly show the need to continue studying the biological properties of the essential oils from aromatic plants.
Acknowledgments
The authors would like to thank Agencia Cordoba Ciencias S.E., SECYT-UNC, and SECYT-Universidad Nacional de Río Cuarto for funding. Fellowholder: Maria de las Mercedes Oliva (Agencia Córdoba Ciencia).
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