The Mediterranean Red Alga Asparagopsis: A Source of Compounds against Leishmania

Crude extracts and column fractions from the red algae Asparagopsis taxiformis and A. armata from the Strait of Messina (Italy) were screened for the production of antimicrobial compounds. Extracts from both species revealed remarkable antiprotozoal activity against Leishmania, revealing such algae as a great source of natural antiprotozoal products.


Introduction
The interest in marine organisms as a potential and promising source of pharmaceutical agents has increased during the last years [1][2][3][4]. To date, many chemically unique compounds of marine origin with various biological activities have been isolated, and some of them are under investigation and are being used to develop new pharmaceuticals [5].

OPEN ACCESS
This genus includes tropical-subtropical red seaweeds, with diplohaplontic life cycle and an heteromorphic tetrasporophyte known as the 'Falkenbergia' stage. To date the genus is represented in the Mediterranean Sea by two species, A. armata and A. taxiformis. A. armata, a temperate distributed species, is considered a Lessepsian immigrant, first reported from the Algerian coasts in 1923 [10]. A. taxiformis, a tropical to warm temperate species, is considered a pre-Lessepsian immigrant or native in the eastern Mediterranean [11], since the first record in the Mediterranean Sea was given in Egypt [12]. Both taxa exhibit a strong invasive behaviour and are included in the list of the "Worst invasive alien species threatening biodiversity in Europe" (EEA 2007) and also in the list of the 100 "Worst Invasives in the Mediterranean Sea" [13].
A. taxiformis and A. armata, as well as other species of the family Bonnemaisoniaceae are well known as sources of halogenated compounds [8,14] with strong antifungal and antibiotic activity [8,15]. Dichloromethane extracts obtained from A. armata exhibited a strong activity against fish pathogenic bacteria [16].
In general, the production of biologically-active metabolites is inherently linked to an ability to partition compounds into specialised storage structures in order to avoid autotoxicity [17].
Members of the Bonnemaisoniaceae form specialized cells [18][19][20] typically known as vesicle or gland cells [21]. In the tetrasporophyte of A. armata, the halogenated metabolites accumulate as a refractile inclusion inside specialized gland cells and this inclusion is no longer produced when the alga is cultured without bromine [21].
The pungent aroma of these algae is due to an essential oil that is composed mainly of bromoform with smaller amounts of other bromine, chlorine, and iodine-containing methane, ethane, ethanol, acetaldehydes, acetones, 2-acetoxypropanes, propenes, epoxypropanes, acroleins and butenones [14]. The halogenated compounds from Asparagopsis have a wide range of volatility and solubility and, hence, no single method of extraction and isolation can be considered entirely satisfactory [8].
In the present paper we present data on the production of antimicrobial halogenated compounds on crude extracts and column fractions from A. taxiformis and A. armata against Leishmania.

Results and Discussion
Three different solvents with increasing polarity were used for the extraction of freeze-dried seaweed powder. Our observations of the effects of extraction method on bioactivity revealed that the active compounds are probably non polar to moderate polar, since the highest activity was observed in ethanol crude extracts fractioned with hexane:ethyl acetate and ethyl acetate. A series of small molecular volatile halogenated compounds (halomethanes, haloethers, haloacetals) are described as responsible for the antimicrobial action of A. armata [8].
Hexane and dichloromethane crude extracts of A. armata also showed a remarkable inhibition, however, both IC 50 and IC 90 values were low (over 40.00 μg/mL) at the same experimental conditions ( Table 1).
The active fractions obtained from ethanol crude extracts of A. taxiformis were eluted with hexaneethyl acetate and ethyl acetate. IC 50 values were 14.00 μg/mL and 20.00 μg/mL, for both fractions, respectively, and IC 90 were 32.00 μg/mL and 34.00 μg/mL, for both fractions, respectively ( Table 1).
The same moderate polar fractions from A. armata resulted active with IC 50 of 10.00 and 19.00 μg/mL, IC 90 30.00 and 32.00 μg/mL under the same experimental conditions (Table 1).
Pentamidine and amphotericin B were tested as control drugs. Two different inhibition assays were performed. IC 50 values ranged from 0.9 to 1.0 mg/mL and IC 90 s ranged from 1.9 to 4.0 mg/mL for pentamidine, while IC 50 s ranged from 0.18 to 0.19 mg/mL and the IC 90 was 0.32 mg/mL for amphotericin B (Table 2). Leishmaniasis is a disease caused by the protozoa of the Leishmania species and it has a worldwide distribution, especially in many tropical and sub-tropical countries. It affects as many as 12 million people worldwide, with 1.5-2 million new cases each year. There is increasing awareness that drug treatment can be complicated by variation in the sensitivity of Leishmania species to drugs, variation in pharmacokinetics, and variation in drug-host immune response interaction [26,27].
The LC-MS analysis of the column fraction in ethyl acetate from ethanol crude extracts of A. taxiformis revealed two peaks of nearly the same intensity at m/z 303.1 and 305.1 [M+H], which indicates presence of one bromine atom. Due to the small quantities of extracts and fractions, further characterization of this compound was not possible. The presence of a small molecular weight brominated molecule in the active fraction confirms that the lipophilic halogenated compounds are truly the metabolites responsible for potent antimicrobial activity of this extract.

Experimental Section
Plants of A. taxiformis and A. armata were collected from the Strait of Messina (Italy), respectively at Torre Faro, Messina and Villa San Giovanni, Reggio Calabria in May 2008. Fresh plants were washed in sterile sea water and manually cleaned of epiphytes. Lyophilized and powdered plants of A. taxiformis and A. armata (dry weights: 75 g for each species) were extracted using different organic solvents with increasing polarity (hexane, dichloromethane and ethanol) at room temperature. Extracts were dried with a Rotavapor® at low temperature (35 °C) to prevent volatile compounds from evaporation.
In vitro antimicrobial susceptibility assays were performed on Leishmania donovani promastigotes cultures (2 × 10 6 cell/mL). A transgenic cell line of L. donovani promastigotes showing stable expression of luciferase was used as the test organism. The plates were incubated at 26 °C for 72 h, and growth of Leishmania promastigotes was determined by the Alamar blue assay [28]. Pentamidine and amphotericine B were tested as the standard antileishmanial agents. Microbiological assays were performed at the Microbiology laboratory of National Center For Natural Products Research of the University of Mississippi.
The hexane and dichloromethane extracts were not further fractionated because of limited amount of materials. Ethanol extracts of A. taxiformis and A. armata were submitted to fractionation using Si gel vacuum liquid chromatography eluted in order with hexane, hexane-ethyl acetate (1:1), ethyl acetate, ethyl acetate-methanol (1:1), methanol, water. Fractions were tested in antimicrobial assays.
Fractionation and isolation of compounds were further performed using HPLC, with a normal phase Silica gel column (10 mm) as stationary phase and gradient of two solvents, hexane and isopropanol, as mobile phase. Each fraction was dried in vacuum and 1 H-NMR spectra in CDCl 3 was recorded on a Bruker BioSpin instrument operating at 400 MHz. LC-MS analysis for each sample was carried out with a micrOTOF ESI-TOF MS.

Conclusions
Red algae of the genus Asparagopsis are well known as sources of halogenated compounds with strong antifungal and antibacterial activity [8,[14][15][16], but, as far as we know, there are no published data on their activity against any protozoa. According to our results, A. armata and A. taxiformis revealed high potential as source of natural products with antiprotozoal activity in vitro. This first report of the effectiveness of Asparagopsis against Leishmania represents a challenge to encourage explorative research on such topic. Asparagopsis species merit further studies both with the aim of isolating their active metabolites on larger scale and for assaying culture methods for supplying algal biomass for industry.