Antileishmanial activity in vitro and in vivo of ... - Springer Link

6 downloads 0 Views 116KB Size Report
May 2, 2008 - Abstract In the search of new antileishmanial drugs from marine resources, we have investigated Actinopyga lecanora, a coral reef sea ...
Parasitol Res (2008) 103:351–354 DOI 10.1007/s00436-008-0979-3

ORIGINAL PAPER

Antileishmanial activity in vitro and in vivo of constituents of sea cucumber Actinopyga lecanora Nasib Singh & Rajesh Kumar & Swati Gupta & Anuradha Dube & V. Lakshmi

Received: 14 September 2007 / Accepted: 31 March 2008 / Published online: 2 May 2008 # Springer-Verlag 2008

Abstract In the search of new antileishmanial drugs from marine resources, we have investigated Actinopyga lecanora, a coral reef sea cucumber, for its in vitro and in vivo activities. Methanol extract and n-butanol fraction of A. lecanora exhibited excellent Leishmania donovani inhibition. Among the two glycosides isolated from n-butanol fraction, holothurin B (2) displayed excellent in vitro and moderate in vivo leishmanicidal activity, whereas holothurin A (1) revealed only moderate action. These findings provide an important marine lead toward the development of new antileishmanial drugs and necessitate the further exploration of rich and diverse marine resources for more potent bioactive molecules.

Leishmaniasis is one of the major tropical parasitic disease affecting more than 12 million people in 88 countries (http://www.who.int/tdr/diseases/leish/default.htm). Leishmaniasis is caused by Leishmania spp., which is a flagellate protozoan parasite transmitted by phlebotomine sandfly. Its manifestations range from a self-healing cutaneous form to fatal visceral leishmaniasis (VL), also known as kala-azar in India. There is no vaccine available against VL. Drug resistance, variable efficacy, toxicity, parenteral administraN. Singh : S. Gupta : A. Dube Parasitology Division, Central Drug Research Institute, Chattar Manzil Palace, Lucknow 226001, India R. Kumar : V. Lakshmi (*) Medicinal and Process Chemistry Division, Central Drug Research Institute, Chattar Manzil Palace, Lucknow 226001, India e-mail: [email protected]

tion, and requirement for long courses of administration are the main drawbacks of current antileishmanial drugs (Croft and Coombs 2003; Croft et al. 2006). There is an urgent need for new drugs for the treatment of these diseases, which mainly affect people in developing countries. With the recent emphasis of the World Health Organization (WHO) on the development of antileishmanial agents from natural products, urgent search for potent drug candidates derived from Indian marine organisms has been initiated. Many studies on natural products extracted from marine organisms have led to the discovery of substances with potential pharmacological activity (Faulkner 2001; Haefner 2003; Dube et al. 2007). In this direction, we have explored Actinopyga lecanora, a coral reef sea cucumber, for its potential as an antileishmanial drug candidate. We report herein the in vitro and in vivo antileishmanial activity of crude extract, fractions, and two triterpene glycosides of A. lecanora against Leishmania donovani.

Materials and methods Extraction, fractionation, and structure elucidation of pure compounds The marine animal freshly collected (10 kg) was chopped into small pieces filled in glass percolators and extracted with methanol and afforded extract (60.0 g). Crude methanol extract (45.0 g) was fractionated into ethyl acetate (10.5 g) and n-butanol (20.8 g) soluble fractions. Since activity was localized in a n-butanol soluble fraction, therefore the n-butanol fraction was subjected to column chromatography over flash silica gel (230–400 mesh), eluting with a gradient elution of CHCl3/MeOH/H2O (90:9.5:0.5 to 88:11.25:0.75) afforded a pure compound

352

Parasitol Res (2008) 103:351–354

(1) (400 mg) and compound (2) (50 mg) by repeated column chromatography. On characterization with the help of physicochemical techniques, these two compounds were identified as holothurin A (1) (Kitagawa et al. 1981a) and holothurin B (2) (Kitagawa et al. 1981b), respectively, by comparing their spectroscopic data with those reported in the literature (Table 1). Antileishmanial activity against promastigotes Green fluorescent protein-transfected promastigotes (5×105 cells per ml) were put into 48-well plates, and different dilutions of test samples and standard drug were administered in triplicate. After 96 h, treated cells were analyzed by the flow cytometric method as described previously by Singh and Dube (2004), Tripathi et al. (2006), and Dube et al. (2007). Antileishmanial activity against intracellular amastigotes Mouse macrophage cell line J774 was maintained in Dulbecco’s modified Eagle’s medium (Sigma, USA) supplemented with 10% heat-inactivated Fetal bovine serum at 37°C in 5% CO2. Cells were cultured in 16-well chamber slides (Nunc, USA) to a cell density of 105 cells per well Table 1 Structure and nomenclature of compounds

Compound 1

Chemical name Holothurin A

2

Holothurin B

Structure

R=H

and infected with late log-phase promastigotes at a multiplicity of infection of 10:1 (parasite/macrophage) and incubated at 37°C in 5% CO2 for 8–12 h. Nonphagocytosed promastigotes were removed by washing, and the drug dilutions were administered. After 72 h, chamber slides were fixed in absolute methanol, stained with 10% Giemsa, and examined under an oil immersion objective of the light microscope. At least 100 macrophages were counted per well for calculating the percentage of infected macrophages, and the percent inhibition was calculated (Dube et al. 2005; Tripathi et al. 2006; Table 2). In vivo leishmanicidal efficacy against experimental VL in golden hamsters L. donovani-infected hamsters were administered with different doses of test samples by oral route. Miltefosine, the reference drug, was administered orally at a dose of 40 mg/kg×5. Splenic parasite loads in treated and untreated animals were determined on day 7 post-treatment by performing splenic biopsies (Lakshmi et al. 2007). Impression smears were made on glass slides, air dried, fixed in absolute methanol, and stained with 10% Giemsa for 30 min. Slides were counted for the number of amastigotes under the oil immersion objective (100×) of a phase-contrast micro-

Parasitol Res (2008) 103:351–354

353

Table 2 In vitro antileishmanial activity of methanol extract, fractions, and triterpene glycosides of A. lecanora Test sample/drug

Activity against promastigotes

Activity against intracellular amastigotes

Doses (μg/ml)

Doses (μg/ml)

Methanol extract

Percent inhibition (mean±SD)

100 50 100 50 100 50 100 50 100 50 10

Ethyl acetate soluble fraction n-Butanol soluble fraction Holothurin A (1) Holothurin B (2) Miltefosineb

88. 5±8.4 56.1±7.8 21.0±7.1 NIa 98.5±9.6 68.6±8.2 73.2±6.8 40.4±7.1 82.545±5.511.6 47.345±5.56.5 100

Percent inhibition (mean±SD)

100 50 100 50 100 50 100 50 100 50 10

72.45±5 47.6±6 17.4±4 NI 76.4±3.7 58.2±5 65.8±6 45±5.5 7845±5.510.2 57.645±5.58.4 100

Data (means±SD) are representative of three separate experiments. NI No inhibition b Miltefosine used as standard antileishmanial drug a

scope (Singh et al. 2007). Percent parasite inhibition in treated animals was calculated using the formula % inhibition ¼

AT  100 ITTI

where AT is the actual number of amastigotes per 100 spleen cell nuclei in treated animals, IT is the initial number of amastigotes per 100 spleen cell nuclei in treated animals, and TI is the times increase in untreated control animals.

Results and discussion As a part of our screening program in search for bioactive molecules from the marine organisms of the Indian Ocean,

we have investigated the sea cucumber A. lecanora for antileishmanial activity. The methanol extract exhibited 88.5±8.4% and 72.4±6% inhibition of promastigotes and intracellular amastigotes, respectively, at 100 μg/ml concentration. At a lower dose of 50 μg/ml, it demonstrated 56.1% and 47.6% inhibition of promastigotes and intracellular amastigotes. Among the fractions, n-butanol was most potent resulting in 98.5±9.6% and 68.6±8.2% inhibition of promastigotes and 76.4±3.7% and 58.2±5% inhibition of amastigotes at 100- and 50-μg/ml concentrations. Ethyl acetate fraction was inactive against both stages of the parasite. Therapeutic treatment of L. donovani-infected hamsters with methanol extract resulted in 69.4±4.2% (25±5.2 amastigotes per 100 nuclei, expressed as parasite

Parasite burden (No. of amastigotes/100 cell nuclei)

Parasite burden (No. of amastigotes/100 cell nuclei)

100 100

80

60

40

20

0

80

** P < 0.005 60

40 Holothurin A Holothurin B

20

0 500

250

Methanol extract

500

250

n-Butanol fraction

500

250

Milt

Cont

Ethyl acetate fraction

Fig. 1 Antileishmanial activity of methanol extract and fractions of A. lecanora against L. donovani infection in hamsters. Results are expressed as mean±SD of two independent experiments. The number of animals in each group per replicate was three

*** P < 0.0001

50 mg

100 mg

Doses (mg/kg/day× 5)

Fig. 2 In vivo antileishmanial activity of pure compounds isolated from the n-butanol fraction of A. lecanora against L. donovani infection in hamsters. Results are expressed as mean±SD of two independent experiments. The number of animals in each group per replicate was three

354

burden) and 32.1±8.6% inhibition at dose levels of 500 and 250 mg kg−1 day−1 ×5, respectively. The treatment with the n-butanol fraction at a 500-mg/kg dose reduced the parasite burden to 26±4.8 compared to 85±9.6 amastigotes per 100 cell nuclei in untreated group. The ethyl acetate fraction was totally inactive. Miltefosine, the reference drug, exhibited 93.9±2.6% inhibition at a 40-mg kg−1 day−1 ×5 dose (Fig. 1). Among the pure compounds isolated from the bioassayguided fractionation of n-butanol, compound (1) showed moderate activity both in vitro and in vivo. At 100 μg/ml, it demonstrated 73.2±6.8% and 65.8±6% inhibition of promastigotes and intracellular amastigotes, respectively. Inhibitory activity was lower at decreasing concentrations. Against established infection in hamsters, it exhibited 44.6±10.6% inhibition at a dose of 100 mg kg−1 day−1 ×5, whereas it was inactive at lower doses. Compound (2) exhibited superior antileishmanial activity than compound (1). It inhibited the growth of promastigotes to the tune of 82.5±11.6 and 47.3± 6.5 at 100- and 50-μg/ml concentrations, respectively. Similarly against intracellular amastigotes, it imparted 57.6± 8.4% and 78±10.2% inhibition at 50 and 100 μg/ml, respectively. In L. donovani-infected hamsters, compound (2) revealed better efficacy (71.5±12.8%; P