Chemical composition , toxicity and larvicidal and antifungal activities of Persea americana ( avocado ) seed extracts

The present study had the aim of testing the hexane and methanol extracts of avocado seeds, in order to determine their toxicity towards Artemia salina, evaluate their larvicidal activity towards Aedes aegypti and investigate their in vitro antifungal potential against strains of Candida spp, Cryptococcus neoformans and Malassezia pachydermatis through the microdilution technique. In toxicity tests on Artemia salina, the hexane and methanol extracts from avocado seeds showed LC 50 values of 2.37 and 24.13mg mL respectively. Against Aedes aegypti larvae, the LC 50 results obtained were 16.7mg mL for hexane extract and 8.87mg mL for methanol extract from avocado seeds. The extracts tested were also active against all the yeast strains tested in vitro, with differing results such that the minimum inhibitory concentration of the hexane extract ranged from 0.625 to 1.25mg L-, from 0.312 to 0.625mg mL and from 0.031 to 0.625mg mL, for the strains of Candida spp, Cryptococcus neoformans and Malassezia pachydermatis, respectively. The minimal inhibitory concentration for the methanol extract ranged from 0.125 to 0.625mg mL, from 0.08 to 0.156mg mL and from 0.312 to 0.625mg mL, for the strains of Candida spp., Cryptococcus neoformans and Malassezia pachydermatis, respectively. Key-words: Avocado seeds. Artemia salina. Aedes aegypti. Yeasts.

Persea americana Mill.(Lauraceae) is a plant from Central America (Mexico, Guatemala, Antilles), but it has shown easy adaptation to other tropical regions.Its fruits are commonly known as avocados and have an olive-green peel and thick pale yellow pulp that is rich in vegetable oils and appreciated for its sensory attributes.There is a global tendency towards fruit processing and, following such industrial processes, byproducts like avocado seeds are normally discarded.However, these byproducts from industrialization can cause ecological problems like increased numbers of insects and rodents.In addition, there are economic losses due to the high cost of transporting these byproducts to disposal areas 6 .Thus, studies to investigate the benefits of these byproducts as sources of food supplements or medicinal products are needed 21 .
From the perspective of the use of avocado seeds as sources of phytotherapeutic agents, they have traditionally been used to treat mycoses and parasitic infections.Furthermore, avocado seed preparations are known to have local anesthetic effects that decrease muscle pain 21 .
Previous phytochemical studies on avocado seeds have identified various classes of natural products such as phytosterols, triterpenes, fatty acids, furanoic acids, flavonol dimers, proanthocyanidins and abscisic acid.Some of them are related to antifungal activity 1 and larvicidal effect 15 .
In the present study, the methanol and hexane extracts of avocado seeds were tested with regard to their toxicity, using the microcrustaceus Artemia salina and third-stage Aedes aegypti larvae.In addition, their antifungal potential using the microdilution method was evaluated against Candida spp, Cryptococcus neoformans and Malassezia pachydermatis.

Botanical material.
The seeds used in this study were obtained from a food industry located in Fortaleza, Ceará, Brazil, during August and September 2005.The cultivars were located in the State of Ceará, and especially within the metropolitan region of Fortaleza.
Sample preparation.The seeds had previously been separated from the pulp, dried in an oven at 50ºC and ground into powder in a laboratory mill, and were kept wrapped in filter paper cartridges.The extracts were obtained by heat extraction using the reflux method (Soxhlet), using hexane and methanol separately as solvents for six hours.They were named hexane extract of avocado seeds (HEAS) and methanol extract of avocado seeds (MEAS), respectively.The extracts were concentrated in a rotary evaporator, under reduced pressure (60rpm at 80°C).
Toxicity tests using Artemia salina.These assays were developed as described by Krishnaraju et al 10 , with modifications.Thus, 50mg of hexane and methanol extracts from avocado seeds were weighed and dissolved in 500μl of dimethyl sulfoxide (DMSO).Seawater was added up to a final volume of 50mL.Different concentrations were obtained through dilutions, thus resulting in final concentrations of 1, 10, 100 and 1,000mg mL -1 .The assays were performed in triplicate, and a control assay was performed using only DMSO and seawater.Artemia salina larvae were added and, after 24h of contact, the survivors were counted and the mean lethal concentration (LC 50 ) was determined.
Larvicidal activity against Aedes aegypti.To detect larvicidal activity, third-stage Aedes aegypti larvae were used.Different masses of extract were dissolved in 300μl of DMSO, and 19.7mL of water were added to obtain solutions, thus resulting in concentrations of 500, 250, 100 and 50mg mL -1 .These were tested against 50 Aedes aegypti larvae.This assay was performed in triplicate at 28 ± 1ºC with relative air humidity of 80 ± 5% and light/dark cycling every 12h.All the experiments were followed by performing a control series with the same number of larvae in DMSO and water.Mortality was observed 24h after exposure.In accordance with the mortality found from the initial concentrations, intermediate concentrations were tested in order to determine the LC 50 of the samples 3 .
Fungal strains.The fungal strains were obtained from the Specialized Medical Mycology Center, Federal University of Ceará, Brazil.Candida spp and Malassezia pachydermatis strains were isolated from healthy dogs and Cryptococcus neoformans from pigeon feces.
Antifungal activity.The sensitivity of antifungal agents was evaluated using microdilution in Roswell Park Memorial Institute (RPMI) 1640 broth, in accordance with the methodology suggested by CLSI (Clinical Laboratory Standard Institute) for Candida spp and Cryptococcus neoformans strains.The inoculum of the yeast was adjusted to obtain 2.5 to 5 x 10 3 cells/mL.Negative and positive controls were added to each 96-well plate, and each yeast was also tested with DMSO and its dilutions.The microplates were incubated at 32ºC for 48h.The test readings were done by visual comparison.For Malassezia pachydermatis yeast, the method of microdilution in RPMI 1640 was used, supplemented with (per liter): 20g of glucose, 4g of ox bile, 1mL of glycerol, 0.5g of glycerol monostearate and 0.4mL of Tween 20 22 .The inoculum of the yeast was adjusted to obtain a 2.5 to 5 x 10 3 cells/mL.Negative and positive controls were added to each 96-well plate, and each yeast was also tested with DMSO and its dilutions.The microplates were incubated at 32ºC for 48h.According to Prado et al 18 , for minimum inhibitory concentration (MIC) determination of Malassezia pachydermatis, 1μl from each well needs to be transferred to and distributed on Petri dishes with potato-dextrose agar, after this period.These dishes were incubated at 32ºC and, after 96h, the fungal colonies were counted.The MIC was taken to be the lowest concentration capable of inhibiting 100% of the growth of each yeast, with its respective positive control as the reference 12 .
Phytochemical screening and isolation of compounds.The phytochemical screening was developed using the methodology described by Matos 14 , to determine the presence of flavonoids, anthocyanins, saponins, tannins, sterols, triterpenoids, fixed acids and alkaloids.The methyl esters were analyzed using the transmethylation technique followed by gas chromatography/mass spectrometry (GC/MS).The hexane extract was analyzed in a silica gel chromatographic column, using hexane, chloroform, ethyl acetate and methanol as eluents, in mixtures of increasing polarity.The ethyl acetate fraction was subjected to subsequent silica gel column chromatography following the same procedure as above.The fractions obtained were monitored chemically using thin layer chromatography (TLC), to achieve isolation of two compounds.The chemical structures of the compounds were determined by spectroscopic methods ( 1 H and 13 C NMR) and comparison with authentic samples.

RESULTS
The HEAS and MEAS showed LC 50 values of 2.37 and 24.13mg mL -1 respectively, in the toxicity assays using Artemia salina.In the larvicidal assays, the extracts showed activity against Aedes aegypti, with LC 50 of 8.87mg mL -1 for HEAS and 16.7mg mL -1 for MEAS (Table 1).The hexane and methanol extracts were active against all the yeasts tested by the microdilution method, and these results are reported in Table 2.
HEAS: hexane extract of avocado seeds, MEAS: methanol extract of avocado seeds, MIC: minimum inhibitory concentration, *represents the number of strains for the MIC indicated.

FIgURE 1
Molecular structure of the compounds identified from the hexane extract of avocado seeds.

DISCUSSION
In the present study, the various extracts were submitted to toxicity assays using Artemia salina.They were considered bioactive, since they showed an LC 50 lower than 1,000mg mL -1 1 5 .The hexane extract from avocado seeds showed the highest toxicity to Artemia salina (LC 50 of 2.37mg mL -1 ).
Determination of the toxicity to Artemia salina has been used efficiently to analyze the biological potential of plant extracts.Several natural products, especially substances with antifungal, antiviral, antibacterial and trypanosomicidal activity, have been tested through this assay, showing a significant relationship.Parra et al 18 showed that it is a fast, simple, practical and low cost assay.Moreover, it avoids unnecessary use of animals in laboratories, because the LC 50 and LD 50 values show a good relationship between in vivo tests, with a rate of r = 0.85.In addition, this method requires small quantities of the samples that can be tested on a large scale.
This study also investigated the antifungal potential of avocado seed extracts against Candida spp, Cryptococcus neoformans and Malassezia pachydermatis.These yeasts are of great importance for human and veterinary medicine 2 .Candida species are one of the main agents responsible for fungal infections in hospitals.Recently, cases of antifungal resistance within this fungal group have been described.The avocado seed extracts induced inhibition of the growth of Candida species tested, producing MIC for HEAS ranging from 0.312 to 1.25mg mL -1 and for MEAS from 0.125 to 0.625mg mL -1 .The HEAS MIC values ranged from 0.031 to 0.625mg mL -1 and, for MEAS, from 0.321 to 0.625mg mL -1 for Malassezia pachydermatis strains.Malassezia pachydermatis has high veterinary importance because of its intense pathological activity on carnivorous animals, but has also been related to fungemia cases among neonates 4 .
The extracts demonstrated lower concentration ranges against Cryptococcus neoformans strains, with values from 0.312mg mL -1 (n=5) to 0.625mg mL -1 (n=1) for HEAS, and from 0.08mg mL -1 (n=6) to 0.156mg mL -1 (n=1) for MEAS.Cryptococcus neoformans is a yeast of essentially opportunist action that is responsible for cryptococcosis cases among HIV-positive patients 13 .The MIC values obtained against Cryptococcus neoformans strains were lower than the values against Candida spp and Malassezia pachydermatis strains.

TABLE 1
Lethal concentration (LC 50 ) of avocado seed extracts against Artemia salina and Aedes aegypti larvae.