Anti-Leishmania Activity of Osthole

Extraction and purification of osthole from Prangos asperula Boiss.

P. asperula Boiss. was collected in April - June 2013 from Dena mountains, environs of Yasouj (West of Iran), at an altitude of 2500 m above the sea level. The plant was identified by Azizollah Jafari, Department of Botany, Yasouj University. Osthole was isolated via n-hexane soxhlet extraction of 100 g air dried fruits. The extract was cooled at 4–5°C following evaporation of the solvent to form a semi pure yellow mass, later washed with chilled n-hexane for several times to enhance the purification. Eventually, the sample was recrystallized until pure osthole crystalline was formed.[13] A voucher specimen of the plant is retained at the herbarium of Department of Pharmacognosy, School of Pharmacy, Isfahan University of Medical Sciences, Iran (1126).

Parasite culture

L. major (MRHO/IR/75/ER) has been isolated from the previously infected BALB/c mice spleens and kept cryopreserved at −80°C. The promastigotes were warmed up to 25°C and incubated for 3–5 days in N.N.N medium; subsequently, passaged at 25°C in RPMI 1640 medium (PAA, Australia), supplemented with 10% inactivated fetal bovine serum (Sigma-Aldrich, US), 100 U/ml penicillin, and 100 μg/ml streptomycin.[14]

Macrophage culture

Murine macrophage cell lines (J774 A.1) were purchased from Pasteur Institute, Tehran, Iran. The cryopreserved cell lines were melted, immediately washed with phosphate-buffered saline (PBS), and cultured in RPMI 1640 supplemented with 20% fetal bovine serum and 100 μg/ml streptomycin, subsequently, incubated in 5% CO₂ at 37°C. By the time the macrophages developed pseudopodia and adhered to the bottom of the plates, they were harvested utilizing a cell scraper and washed by PBS (centrifuged at 1500 rpm for 5 min), and seeded in to 6 well culture plates at concentration of 2 × 10⁶ macrophages/well.[15]

Evaluation of anti-amastigote activity

Promastigotes in the stationary phase of growth were centrifuged 3000 rpm for 10 min at room temperature, then resuspended and washed with medium. To infect the macrophages, parasites at the ratio of seven promastigotes per macrophage were introduced to the cultures in which the fresh medium had been replaced previously. The infected cultures were incubated at 33°C for 6 h, and to complete the phagocytosis process at 37°C for 24 h. Subsequently, they were washed with medium to remove the unphagocytosed parasites. Each well was daily observed using an invert microscope to determine the number of amastigotes and intensity of the infection. By the time 80% of the macrophages were infected, osthole with serial concentrations (5–50 μg/ml) was added (osthole was dissolved in dimethyl sulfoxide [DMSO] and diluted by complete medium). DMSO treated and untreated infected macrophages were used as negative controls and 100 μg/ml meglumine antimoniate, MA-treated (Glucantime^®, Aventis, France) infected macrophages was used as positive control.

The cells were fixed with methanol and stained with Giemsa 12, 24, 48, and 72 h posttreatment. Anti-amastigote activity was evaluated by numeration of the amastigotes in 100 macrophages. Each assay was performed in triplicate. The IC₅₀ value was determined using a logarithmic dose – response regression curve.[16]

Animals

The in vivo procedures of this experimental study were in compliance with guidelines of Isfahan University of Medical Sciences (Isfahan, Iran) to keep and use the laboratory animals in accordance with the Animal Ethics Committee.

The animals used in this research provided by the Center for Research and Training in Skin Diseases and Leprosy, Tehran, Iran, were 6–8 week-old-mice, 20 ± 5 g weight female BALB/c type.

The animals were housed in standard cages with access to water and standard food at 21°C ± 2°C, and 40–50% humidity conditions in a colony room under 12:12 h light/dark cycle.[17]

Infecting BALB/c mice by injection of Leishmania major

CL lesions were induced at the base of tail via subcutaneous injection of 10⁷ viable promastigotes in 0.1 ml PBS.[18]

Treatment of infected animals

Four weeks later when CL lesions were developed, mice with mean lesion size ≈2.3 ± 0.2 mm were selected and divided into four groups of eight (A, B, C, and D). Mice of group (A) were treated topically with 0.2% osthole[19] (osthole dissolved in 1 ml DMSO diluted by xanthan gel up to 10 ml), group (B) received 100 mg/kg Glucantime^® intralesionally, and group (C) was treated with 10% (v/v) DMSO-xanthan gel. The treatment was performed daily for 14 consecutive days, and mice of group (D) were left untreated.[19]

Measurement of lesion size

Before, during, and 14 days after the treatment period the horizontal and vertical diameters (mm) of lesions were measured using calipers every 4 days. Mean lesion size in each animal was determined according to the formula S = (D + d)/2.[17]

Statistical analysis

In this study, all the results are represented in means ± standard deviation. The statistical data analysis was done utilizing independent samples t-test for the in vitro study and paired samples t-test for the in vivo study. P < 0.05 is considered statistically significant.

In vitro study

The results obtained from numeration of intracellular amastigotes demonstrated that osthole decreased the viability of amastigotes in J774.A1 macrophages in a dose-dependent manner with IC₅₀ value of 14.95 μg/ml after 72 h [Figure 1]. Anti-amastigote activity against L. major was found similar to Glucantime^® at concentrations ≥ 20 μg/ml (P > 0.05). No significant difference in viability of amastigotes and macrophages in negative controls were observed (P > 0.05). Osthole affected cell viability of J774.A1 macrophages at concentrations ≥30 μg/ml.

Figure 1

Mean number of intracellular amastigotes in negative controls, increasing concentrations of osthole, and positive control in 12, 24, 48, and 72 h. Number of amastigotes is significantly decreased at concentrations ≥20 μg/ml (P < 0.05)

In vivo study

BALB/c mice infected with promastigotes of L. major developed progressive lesions 4 weeks after the injection of inoculum. Mice of groups C and D (DMSO-xanthan treated and untreated) developed larger lesions, (5.19 ± 0.19 mm) and (5.11 ± 0.28 mm), respectively. Mean lesion size of group A (osthole treated) was found to be significantly less compared to groups C and D (P < 0.05; 3.9 ± 0.32 mm). Unlike group B (Glucantime^® treated) subsidence of lesion size did not occur in group A [Figure 2].

Figure 2

Mean lesion size of osthole treated (A) Glucantime^® treated (B), dimethyl sulfoxide-xanthan treated (C) and untreated (D) mice. Lesion size of group (A) is significantly less than groups (C) and (D) (P < 0.05)

Financial support and sponsorship

Isfahan University of Medical Sciences.

Conflicts of interest

There are no conflicts of interest.