Brine shrimp ( Artemia salina Leach ) bioassay of extracts from Lychnophoriopsis candelabrum and different Lychnophora species

Brine shrimp (Artemia salina Leach) bioassay of extracts from different Lychnophora species. The present study aimed to evaluate on Artemia salina the citotoxicity of twenty-two extracts from five species of the genus Lychnophora and one species of the genus Lychnophoriopsis. The extracts solubilized in DMSO and prepared at the final concentrations of 100, 250, 375, 500 and 600 g mL-1 were added to tubes containing Artemia salina nauplii (10 units each) and filled to 5 mL total volume with artificial salt water. Lapachol and 5% DMSO were used as positive and negative controls, respectively. The samples were kept under light and dead larvae were counted after 24 hours of contact. LC50 was calculated by using Probit software. The crude ethanol extracts from five species showed low lethality in the following order: Lychnophora trichocarpha (LC50 = 672.38 g mL-1) > Lychnophora pinaster (LC50 = 678.73 g mL-1) > Lychnophora ericoides (LC50 = 738.09 g mL-1) > Lychnophoriopsis candelabrum (LC50 = 812.57 g mL-1) > Lychnophora passerina (LC50 = 921.78 g mL-1). All tested extracts from L. candelabrum and chloroform extract from L. staavioides showed light toxicity on A. salina. Results indicated that there are substances with potential pharmacological activity in all tested species.


INTRODUCTION
The genus Lychnophora (Asteraceae) is restricted to Brazilian Cerrado (Robinson, 1999).The shoots and roots of a large number of Lychnophora species are widely used in Brazilian folk medicine.They are macerated in water, ethanol, or "cachaça" (sugar cane spirit), administered by oral route or used topically and as anti-inflammatory to treat bruises, Lychnophora species.The present study aimed to evaluate on Artemia salina the citotoxicity of twenty-two extracts from five species of the genus Lychnophora and one species of the genus Lychnophoriopsis.The extracts solubilized in DMSO and prepared at the final concentrations of 100, 250, 375, 500 and 600 g mL -1 were added to tubes containing Artemia salina nauplii (10 units each) and filled to 5 mL total volume with artificial salt water.Lapachol and 5% DMSO were used as positive and negative controls, respectively.The samples were kept under light and dead larvae were counted after 24 hours of contact.LC 50 was calculated by using Probit software.The crude ethanol extracts from five species showed low lethality in the following order: Lychnophora trichocarpha (LC 50 = 672.38g mL -1 ) > Lychnophora pinaster (LC 50 = 678.73g mL -1 ) > Lychnophora ericoides (LC 50 = 738.09g mL -1 ) > Lychnophoriopsis candelabrum (LC 50 = 812.57g mL -1 ) > Lychnophora passerina (LC 50 = 921.78g mL -1 ).All tested extracts from L. candelabrum and chloroform extract from L. staavioides showed light toxicity on A. salina.Results indicated that there are substances with potential pharmacological activity in all tested species.
Artemia salina, commonly known as the brine shrimp, is a small crustacean, which has been the subject of many studies.The brine shrimp lethality assay is considered one of the most useful tools for the preliminary assessment of general toxicity.It has been established as a safe, practical and economic method to determine the bioactivity of plant products (Subhan et al., 2008).The aim of this method is to provide a front-line screen that can be backed up by more specific and more expensive bioassays once the active compounds have been isolated.
Considering that Lychnophora species are used in the crude extract and are able to have phytoconstituents with unknown activities, the present study was conducted to comparatively verify the in vitro cytotoxic activity of ethanol extracts and fractions from five Lychnophora species and one Lychnophoriopsis species.Lychnophoriopsis candelabrum was included in this study because at the beginning of the experiment it was classified as belonging to the genus Lychnophora (Santos et al., 2005).

Cytotoxic activity
The cytotoxicity of extracts was evaluated by Artemia salina lethality test according to the procedure described by Meyer et al. (1982).Encysted eggs of the brine shrimp Artemia salina Leach were incubated in artificial seawater and exposed to a 60-W lamp, pH 8-9.After 48 h, the nauplii (10 units) were added to each set of tubes containing crude ethanol extract dissolved in 5% DMSO andfilled to 5 mL total volume with artificial salt water.The extracts were tested in triplicate at 100, 250, 375, 500 and 600 g mL -1 .Lapachol and 5% DMSO were used as positive and negative controls, respectively.After 24 h, the number of survivors was counted and the percentage of death calculated.The lethal concentration 50% (LC 50 value) and the standard error were calculated by Probit analysis (Finney, 1971).

RESULT AND DISCUSSION
In the present study, brine shrimp lethality assay was carried out to evaluate the cytotoxicity of extracts from different Lychnophora species.Results are shown in Table 1.
According to Nguta et al. (2011), crude extracts with LC 50 values less than 100 g mL -1 are considered highly toxic, those with LC 50 values between 100 g mL -1 and 500 g mL -1 , moderately toxic, the ones with LC 50 values between 500 g mL -1 and 1000 g mL -1 , mildly toxic, and those with LC 50 values above 1000 g mL -1 are considered non-toxic.
Of the twenty-two tested extracts, thirteen had LC 50 between 500 and 1000 g mL -1 , showing weak toxicity.The crude ethanol extracts of five tested species had mild cytotoxicity in the following order: LTE > LPiE > LEE > LCE > LPaE.The ethanol extract from L. staavioides (LSE) showed no cytotoxicity.This result may indicate a good prospect of use of L. staaviodes which showed anti-rheumatic activity (Ferraz Filha et al., 2006).However, further toxicity studies should be performed to verify the non-toxicity of L. staavioides and the actual feasibility of using this species.Every tested L. candelabrum extract (LCE, LC1, LC2 and LC3) showed mild cytotoxicity.Additionally, LS2, LTE, LT2, LE1, LE2 and LPi2 extracts showed higher larvicidal activity.
The brine shrimp lethality assay has shown good correlation with trypanocidal (Zani et al., 1995), antitumor and pesticidal activities (McLaughlin et al., 1998).The trypanocidal activity of extracts and compounds from L. passerina, L. pinaster and L. trichocarpha has already been shown.In previous studies, lychnopholide showed trypanocidal activity against tripomastigote strains of Trypanosoma cruzi (Oliveira et al., 1996).
Although in plant extracts the substances are usually found at low concentrations, the results of this study indicate that thirteen extracts obtained from all tested species may contain substances with potential biological activity.

ACKNOWLEDGMENT
The authors would like to thank FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais, 193/05), CNPQ (Conselho Nacional de Pesquisa) and UFOP (Universidade Federal de Ouro Preto) for financial support.Luciana Souza Guzzo and Zilma Ferraz Filha thank the CAPES and UFOP respectively for their fellowship.
NT -not tested