Chemical composition and toxicity of the essential oil of Aloysia Paláu species (Verbenaceae) from South Brazil

In Brazil there are nine native species of Aloysia which are poorly studied as the chemical compositions and biological activities. Hence, this study describes the chemical composition determinated by GC-MS of essential oil of the five native and one cultivated species of Aloysia occurring in Rio Grande do Sul and evaluates the cytotoxic effects of the essential oils from A. citrodora, A. lycioides and A. dusenii through the bioassay with Artemia salina. A group of species showed 1,8-cineol as a major compound: A. dusenii (16.2 %), A. citrodora (32.8 %), A. lycioides collected in Guaiba (49.5 %) and A. lycioides collected in São Marcos (17.6 %). While species A. polygalifolia and A. virgata had higher proportions of germacrene-D (11.2 % and 12 %, respectively) and A. chamaedryfolia presented spathulenol (15.6 %). The species A. lycioides collected in Rosário do Sul had β-Phellandrene (23.7 %) as the major compound. All the essential oils tested presented a high toxicity against Artemia salina with LC50 values among 48.12 μg mL and 55.96 μg mL.


Introduction
Verbenaceae is a family of about 31 genera and approximately 918 species of herbs, shrubs, or small trees, mainly distributed in tropical and subtropical regions (Stevens, 2001). Among the most important genus are Lippia and Aloysia (Ricco et al, 2010). Aloysia comprises 30 species and is distributed from the South of the United States and Mexico until the north of Patagonia (Siedo, 2006), characterized by shrubby form, aromatic inflorescences, and known primarily for its essential oils (Hernandez et al, 2003). Nine species of Aloysia are native in Brazil, occurring all in the South region, and A. citrodora is found only under cultivation (Reflora, 2016).
Aromatic plants have been used since ancient times for their medicinal properties (Bakkali et al, 2008) and their odors are determined by the presence of essential oils (EOs), a mixture of volatile low-molecular-weight mono-and sesquiterpenes and other isoprenes (Singh et al, 2002). The EOs are involved in various ecological interactions including ones of medicinal properties such as bactericidal, fungicidal and antiviral properties (Chao et al, 2000), besides they may also exhibit cytotoxic activity (Sacchetti et al, 2005).
The brine shrimp lethality bioassay is rapid, simple, easily mastered, inexpensive, and requires small amounts of test material (Ghisalberti, 1993) to predict toxicity (McLaughlin, 1991). Since its introduction, this test has been successively employed to provide a frontline screen backed up by more specific and more sophisticated bioassays (Apu et al, 2010).
The present study reports the chemical composition by GC-MS of the essential oils of five native species of the genus Aloysia distributed in the Rio Grande do Sul -South Brazil and one cultivated species. Moreover, the cytotoxic activity of essential oil of the species A. citrodora, A. lycioides and A. dusenii were evaluated by brine shrimp bioassay.

Plant material
Aerial parts from six species of Aloysia (leaves, flowers, and stem) were collected at seven locations in the Rio Grande do Sul State, Brazil (Table 1). The samples were identified, and voucher specimens were deposited in the Herbarium of the Universidade de Caxias do Sul (HUCS) and Herbarium of the Universidade Federal do Rio Grande do Sul (ICN). The detailed data referent to each species can be found in Table 1. Source: Authors (2021).

Essential oil extraction
After collection, aerial parts of Aloysia species were dried at room temperature, fragmented, and subjected to extraction. Essential oils were obtained by hydrodistillation in a Clevenger apparatus for 1 hour (Agostini et al, 2009).
Anhydrous sodium sulfate was employed to eliminate essential oil humidity. The essential oils were stored in airtight tubes, wrapped in aluminum foil, and stored in the freezer (-20 °C) prior to use.

Essential oil chemical characterization
Chromatographic analysis was performed using a gas chromatograph coupled to a mass spectrometer detector ( (Adams, 2007). The relative percentage of each component was obtained from chromatographic peak areas, assuming the sum of all eluted peaks was 100 %.

Brine-shrimp bioassay
The cytotoxicity bioassay was done according to Meyer's procedure (Meyer et al, 1982) with modifications on the preparation of the samples. The essential oils tested were obtained from A. citrodora, A. lycioides (V) and A. dusenii.
Approximately 1 g brine shrimp eggs (Artemia salina -Flagner Soares de Souza Ind.) was hatched in a rectangular aquarium (10  20 cm) filled with artificial seawater, which was prepared with 1 L beaker of distilled water containing 30 g of commercial salt mixture (Azevedo Bento S.A. Comércio e Indústria). After 48 hours of incubation, the active shrimp (10-15) were collected by pipette. The nauplii were transferred to culture plates with diluted solutions of the essential oils in 1000 µg mL -1 , 500 µg mL -1 , 100 µg mL -1 , 50 µg mL -1 e 20 µg mL -1 with dimethyl sulfoxide (DMSO) 1%. The total volume was adjusted to 1 mL with artificial seawater. Three replications were done for each dose level and control with artificial seawater and DMSO 1%. After 24 hours, the survivors were counted. The absence of movement of nauplii for 5 minutes was regarded as dead. The bioassay was done three times independently, and the LC50 (Lethal Concentration 50) and 95% CI (Confidence Intervals) were calculated using the Probit Analysis with the software IBM SPSS 21.0.

Chemical composition of the essential oils
Essential oils of six species of Aloysia (Table 1) were analyzed for chemical composition by GC-MS. The essential oils yields ranged from 0.2% (mL 100 g -1 of dried leaves) for A. chamaedryfolia to 2.5% for A. citrodora, depending on the species (  Source: Authors (2021).
The main compounds of each EO showed that each species presented a different composition. However, some constituents are conserved in several species, as linalool and ß-caryophyllene, for example (Table 2). Possibly, the conservation of these constituents may have chemotaxonomic significance to maintain similar morphologic and biochemical characteristics, which will determine the biosynthesis of their secondary metabolism (Sousa et al, 2012). For A. lycioides, collections were performed in three different locations and resulted in different chemical compositions. Thereby, these species were separated into two groups: major compound 1,8-cineol (IV and V) and β-Phellandrene (VI). These differences in major compounds found among the A.lycioides from Guaíba and São Marcos and from Rosário do Sul could be associated with the geographical origin of the material, but could also suggest different chemotypes. The species A. citrodora is known for the predominant presence of citral in their chemical composition (Zigadlo et al, 1994). However, our results did not show the presence of this component. These data may suggest a new chemotype for the species. These results are based on a local collection and do not analyze the intraspecific variation. The different chemistry can occur through the influence of environmental conditions and seasonal variations (Ricciardi et al, 2011). On the other hand, the composition of the essential oil of a plant is also genetically determined and usually specific to a particular organ and characteristic for their stage of development, giving rise to chemotypes in plants rich in essential oils. Tavares et al (2005) showed that differences in the composition of different chemotypes of Lippia alba are not only a product of the influence of environmental factors but mainly reflect the genotypic variation of these plants.
There were predominant monoterpenes ( presented a similar quantity of mono-and sesquiterpenes (29.4% and 28,9%, respectively). These classes of terpenes are related to different biological activities (Singh & Sharma 2015) what can explain the popular use of some Aloysia species for medicinal purposes (Santos et al, 2015). Source: Authors (2021).

Cytotoxicity activities
As expected, the degree of lethality was directly proportional to the essential oil concentration (Table 4). The mortality rate of brine shrimp nauplii was drastically increased as the dose level was increased from 20 µg mL -1 to 100 µg mL -1 . Moreover, a 100 % mortality was observed at 500 µg mL -1 and 1000 µg mL -1 dose levels for all essential oil evaluated. More than 80% of the nauplii remained active in control, with DMSO 1%. The value of LC50, calculated from the 24-hour counts, was not different among the essential oils of the species tested.
Therefore, the toxicity was considered high, and LC50 values were from 48.12 µg mL -1 in A. lycioides (V) to 55.96 µg mL -1 in A. citrodora. The similar toxicity of these essential oils can probably be explained by the main constituent, 1.8-cineol, present in all of them. According to Meyer et al (1982) the LC50 value under 1000 µg mL -1 is pharmacologically active and toxic, classifying these as potentially of pharmacological interest as some authors have already related the brine shrimp lethality with the detection of antitumoral compounds in terrestrial plants (Carballo et al, 2002, Mackeen et al, 2000. However, there is no correlation between the degree of toxicity found for brine shrimp and the toxicity to mammalian cells and the brine shrimp test is used as a tool for approaching the real toxicity (Oliva et al, 2007). Oliva et al (2007) showed in their study the low toxicity of the essential oil from Aloysia tomentosa (LC50 968 µg mL -1 ), and nontoxicity of essential oils from Aloysia polystachia (LC50 6459 µg mL -1 ) and Aloysia triphylla (synonym for A. citrodora) (LC50 1279 µg mL -1 ), differently of the high toxicity observed in this work for all species, especially A. citrodora. Nevertheless, the essential oil composition determined by them for A. triphylla (A. citrodora) was different and rich in limonene, citral, spathulenol, and thujone, showing the importance of chemical characterization to study biological activities.

Conclusion
This work is the first study evaluating the chemical composition of the essential oil of six species of Aloysia from the South of Brazil. The species showed different chemical compositions, but some constituents are conserved in several species.
This chemical information can assist in the taxonomy of the genus. Also, the results demonstrated the cytotoxic activity of some Aloysia essential oils, showing that studies like this are essential in the screening for new substances with potential biological activities. Therefore, the following steps should be a more detailed evaluation of the toxicity presented by this essential oil to secure the safety of using these natural products.