Antileishmanial Activity of Selected Turkish Medicinal Plants

Purpose: To determine the in vitro and in vivo anti-leishmanial activities of extracts obtained from Centaurea calolepis, Phlomis lycia, Eryngium thorifolium, Origanum sipyleum and Galium incanum ssp. centrale. Methods: To estimate the cytotoxicity of plant extracts, WST-1 assay was used. Parasite inhibition in the presence of plant extracts (25 - 500 µg/ml) in comparision with control group and reference group (glucantime, 25 µg/ml) at 12 - 72 h were determined in vitro on L. tropica promastigotes. The in vivo leishmanicidal activity of the extracts was evaluated against L. tropica-infected mice with glucantime as reference drug. Results: The chloroform extract of Galium incanum ssp. centrale showed the highest cytotoxicity with IC 50 value of 0.0316 ± 0.005 μg/ml. In vitro parasite inhibition by the plant extracts ranged between 16.7 ± 0.01 % and 100 ± 0.00 % at 25 μg/ml concentration. The methanol extract of Eryngium thorifolium possessed the highest activity on promastigotes of L. tropica with 100 % inhibition at 25 μg/ml. The water and chloroform extracts of C. calolepis and water and methanol extracts of E. thorifolium at a dose of 100 mg/kg reduced parasitaemia in L. tropica infected mice. Conclusion: Parasite viability results suggest that the methanol extract of Eryngium thorifolium, regarded as non-cytotoxic, is a promising candidate drug for treating L. tropica infection.


INTRODUCTION
Leishmaniasis is a vector-borne disease affecting 400 million people around the world.This public health problem is endemic in 88 countries.After malaria, leishmaniasis is the most important parasitic disease caused by protozoan parasite species from Leishmania genus [1].Sodium stilbogluconate and meglumine antimonite are the most favourable leishmanicidal agents.These pharmaceutical agents administered by injection usually cause pain at the site of injection.In addition to stiff joints and gastrointestinal disorders, they have also been known to cause cardiotoxicity and severe hepatic and renal insufficiencies [2].
Natural herbal products have been used in the treatment of infectious disease for many years.The World Health Organisation (WHO) emphasized that plants used in traditional medicine should primarily be investigated against leishmaniasis [1].All over the world, researchers started on this issue and successful results were achieved [3][4][5].
With this knowledge and as part of our outgoing studies on anti-parasitic activities of Turkish medicinal plants [6][7][8], in the present work, antileishmanial efficacies of extracts prepared from Centaurea calolepis Boiss.Phlomis lycia D. Don., Eryngium thorifolium Boiss., Origanum sipyleum L., Galium incanum ssp.centrale Ehrend.were examined on Leishmania tropica promastigote and amastigote forms.In addition to preliminary phytochemical screening analysis, cytotoxic activities of each plant extract were also determined by WST-1 cell proliferation assay.

EXPERIMENTAL Plant material
The aerial parts of Centaurea calolepis Boiss., Phlomis lycia, Eryngium thorifolium, Origanum sipyleum, Galium incanum ssp.centrale were collected from various localities in Turkey.The plant species were identified by Dr. Cenk Durmuskahya (Izmir Katip Celebi University, Faculty of Forestry, Department of Forest Engineering, Balatcik, İzmir Turkey) and voucher specimens were deposited in Department of Pharmacognosy, Faculty of Pharmacy, Ege University, Izmir, Turkey (The collection areas for the plants, voucher numbers, extracts and their antimicrobial or antiprotozoal effects as well as the phytochemical profile of the plants are listed in Table 1).

Preparation of plant extracts
The plant materials were ground into a fine powder after air-drying.The methanol and chloroform extracts were prepared by maceration under stirring for 48 h at 24 °C where the solvent/plant material ratio was 15:1.The water extracts were prepared by 2 % infusion and all the extraction solvents were filtered through Whatman filter paper no.1.The filtrates were evaporated to dryness under reduced pressure in a rotary evaporator at 40 ˚C.The residues were lyophylised and stored in screw capped vials at -20 ˚C until analysed.

Phytochemical analysis of plant extracts
Phytochemical screening tests for plant secondary metabolites such as tannins, terpenoids, saponins, flavonoids and alkaloids were conducted on the plant extracts as described by Trease and Evans [9] and Harborne [10].[22].

Preparation of Leishmania tropica isolates
Following clinical diagnosis by dermatologist at Harran University Hospital, Urfa, L. tropica was isolated from the patients with cutaneous leishmaniasis infection in Sanlıurfa province which is an endemic area for this infection.Skin lesions of the patients were cleansed with 70 % ethanol before sample aspiration.A 26-gauge needle with syringe containing 0.1-0.2ml of sterile saline was then inserted intradermally into the outer border of the lesion.The syringe was rotated and the tissue fluids were gently aspirated and partly smeared on three slides [23].These preparations were stained with Giemsa and examined under alight microscope.
The remaining aspiration material was inoculated immediately in Nicolle-Novy-McNeal (NNN) culture medium.The culture tubes were kept in an incubator at 25 °C for 7 days.L. tropica promastigote observed during microscopy were transferred to flasks containing RPMI-1640 medium (Biochrom AG), 10 % fetal calf serum (FCS), 200 U/ml of penicillin and 0.2 mg/ml of streptomycin and incubated at 25 °C for mass cultivation.Promastigotes were collected by centrifugation at 1500 rpm at 4 ºC for 10 min and washed with sterile saline before analysis.Leishmania promastigotes were kept in liquid nitrogen at -196°C until the analysis.The identification of Leishmania species and isoenzyme analysis were conducted according to the procedure of the World Health Organisation Leishmania Reference Center [1].

In vitro antileishmanial assay
Concentrations of plant extracts (25, 50, 125, 250 and 500 μg/ml) were prepared for in vitro experiments.The extracts were dissolved in dimethyl sulfoxide (DMSO) and diluted in RPMI medium containing 10 % FCS.The final volume was adjusted to 2 ml with RPMI medium for each well of a 24-well microplate.In all experiments, in order not to affect parasite growth rate, mobility or morphology, the final concentration of DMSO was not higher than 0.5 % (v/v) [24].After haemocytometer counting, promastigotes were suspended to yield 1 × 10 6 cells/ml in each well.As a reference drug, a pentavalent antimonial compound, glucantime was prepared in sterile DMSO.The highest concentration of DMSO and RPMI medium were also used for untreated groups.Microplates were incubated at 25 °C.
The number of parasites were counted with a haemocytometer (Thoma slide) under a light microscope in 12 -72 h.[6,25,26].All the in vitro experiments were run in triplicate and the results were expressed as mean percent inhibition in parasite number.

Animals and experimental infection
Female Balb-C mice (20 -25 g, 5 -7 weeks old) were obtained from Ege University Experimental Animals Center.The mice were maintained on standard pelleted diet and water ad libitum.The study was approved by the Animal Ethics Comittee, Ege University.Promastigotes were cultured in RPMI-1640 medium supplemented with 10 % FCS and collected on the 14th day of the culture.Ten milliliters of the culture fluid was centrifuged and a final dilution of 1 × 10 8 promastigotes/ml was prepared.The promastigote solution (15 μl) was injected subcutaneously into the right hind footpads of the mice [6].The development of the lesion was measured weekly with a dial micrometer during the course of infection.The infection was well established and lesions were obvious.

Drug administration
The mice were divided into 13 groups (n = 7).Treatment trials were initiated 30 days after the establishment of infection.Ten extracts prepared from the plant materials (water and chloroform extracts of Centaurea calolepis, Galium incanum ssp.centrale, Phlomis lycia and water and methanol extracts of Eryngium thorifolium and Origanum sipyleum) were administered at a dose of 100 mg/kg.Glucantime was used as a reference drug at a dose of 28 mg/kg [6].As a negative control, sterile saline solution was administered to mouse in the untreated group.A placebo group was included in the study.All injections were administered intralesionally at 15 µl, five times with 3-day intervals.The size of developing lesions on the footpads of the mice was measured with a dial micrometer weekly during the course of infection and expressed as the differences in size between the infected and uninfected footpads.The parasite vitality was also investigated in the established infection.Needle aspiration samples were taken from infected lesions before and after the treatment.The samples were stained with Giemsa and examined under oil immersion with a light microscope to detect amastigotes.On the 60th day of infection, 50 mg tissue samples were taken from the edges of the lesions for biopsy.They were smeared on the slides, stained with Giemsa and examined on the microscope to search for amastigote form of L. tropica.Some part of biopsy samples were inoculated in NNN medium for investigation of the live promastigotes.Touch slides were also prepared from the samples for investigation of the amastigotes by PCR after DNA isolation.

Statistical analysis
The results of assessments were analysed using Student's t-test with Windows SPSS Version 15.0.P > 0.05 was taken as statistically significant.

Phytochemical analysis
The extraction yields in percentages and the results of preliminary phytochemical analysis of screened plants were demonstrated in Table 2.All the extracts except for water extracts of C. calolepis and P. lycia, gave positive results for terpenoids.Water extract of C. calolepis were positive for flavonoids, tannins and anthracenes.The chloroform extract of P. lycia possessed terpenoids and flavonoids.Water and methanol extracts of O. sipyleum and E. thorifolium were positive for terpenoids, flavonoids and tannins.Anthracenes were present in the water extract of E. thorifolium.

Cytotoxicity of plant extracts
The IC 50 values of plant extracts were summarised in Table 3.The chloroform extract of Galium incanum ssp.centrale showed the highest cytotoxic activity at a concentration of 0.03615 ± 0.005 µg/ml.Chloroform extract of Centaurea calolepis possessed greater cytotoxicity against WI-38 cell lines than its water extract with IC 50 values of 12.6 ± 0.611 and 141.21 ± 0.168 μg/ml respectively.Water and chloroform extracts of Phlomis lycia showed activity with an IC 50 value of 139.19 ± 0.185 and 748.58 ± 0.311 μg/ml respectively.Meanwhile, water and methanol extracts of Eryngium thorifolium and water extract of Galium incanum ssp.centrale had proliferative activities on cell lines.

In vitro anti-leishmanial activity
All the plant extracts showed inhibitory activity ranging between 16.70 and 100 % at twelfth hour analysis at 25 μg/ml concentrations (Table 4).Results showed that all the extracts at 500 μg/ml concentration exhibited parasite inhibiton percentages ranging between 61.7 % and 100 % at twelfth hour.The chloroform extract of Centaurea calolepis, Phlomis lycia and Galium incanum ssp.centrale were found to have higher activity than the water extracts of mentioned plants.As shown in Table 4, at twelfth hour, water extract of Centaurea calolepis reached 99.2 % parasite inhibition at a concentration of 500 μg/ml whereas the chloroform extract showed 100 % inhibition at 50 μg/ml concentration at 24th hour.Water extract (500 μg/ml) of P. lycia had 98.4 % inhibition at twelfth hour, whereas 99.4 % parasite inhibition was observed for the chloroform extract of P. lycia at 25 μg/ml.In comparison with the untreated group, except for twelfth hour analysis results of water extract of Galium incanum ssp.centrale and methanol extract of Origanum siypleum (p > 0.05), all the extracts showed significant differences (p < 0.01).Methanol extract of Eryngium thorifolium with 100 % parasite inhibition at 25 μg/ml concentration possessed highest activity on promastigotes of L. tropica.

In vivo antileishmanial activity
In the in vivo anti-leishmanial activity assays of plant extracts, the average measurement of the lesions on the footpads of mice in infected but untreated group reached 3.32 ± 0.01 mm (Figure 1) whereas the reference glucantime group had an average of 1.71 ± 0.01 mm lesion size at the end of 8th week.All the extracts, except for water and chloroform extracts of G. incanum and chloroform extract of P. lycia, significantly decreased the lesion sizes of mice in treatment groups (Table 5).At the end of the 8th week, the mice in placebo group had 1.34 ± 0.01 mm average footpad size difference whereas the untreated and glucantime group had 3.32 ± 0.01 and 1.71 ± 0.01 mm respectively.The mice receiving water and chloroform extract of C. calolepis had 1.82 ± 0.02 and 1.79 ± 0.01 mm average footpad size respectively.The average footpad sizes observed for the mice treated with E. thorifolium water and methanol extracts were 1.79 ± 0.01 mm and 1.76 ± 0.01 mm respectively.It is evident from the results that the water and chloroform extracts of C. calolepis and water and methanol extracts of E. thorifolium exhibited in vivo activity against L. tropica infected mice whereas the rest of studied plant extracts were found to have insignificant activity as compared to untreated group (p > 0.05).Amastigotes (Figure 2) and promastigotes (Figure 3) were present in the samples taken from the lesion of mice treated with extracts of G. incanum, O. sipyleum and P. lycia.The parasites in the aspiration lesion samples of the group of mice treated with water extract of E. thorifolium and chloroform and water extract of C. calolepis were found to lose vitality on the 60th day of infection.As shown in Table 5, amastigotes were present in the smear preparations of samples taken from mice treated with methanol extract of E. thorifolium, whereas no promastigotes were observed in the NNN medium..00± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 24 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 48-72 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Glucantime 12 100.0± 0.00 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00 24 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00 48-72 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00 100.0 ± 0.00 *Results are expressed as mean ± SD (p < 0.05); **No significant difference compared to untreated group (p>0.05)

DISCUSSION
In recent years, plant-derived products are gaining increasing scientific attention in the ongoing search for better leishmanicidal compounds [27].Some researchers looking for new alternatives for the treatment of leishmaniasis have focused on microorganisms and plants [28].Faramea guainensis was reported to have significant in vivo leishmanicidal activity on Leishmania amazoensis [29].In India, L. donovani-infected mice were successfully treated with ethanolic extracts of Alstonia scholaris and Swertia chirata at a dose of 1 g/kg/day [30].In a study conducted in Spain on antileishmanial activities of 12 plant species, Inula montana, Bupleurum rigidum and Scrophularia scorodonia were found to have promising potential [31].In another study in Colombia, Annona muricata showed higher antileishmanial activity on L. brazielensis and L. paramensis than the reference drug, glucantime [32].However, there are few studies in literature reporting the activity of plant extracts and plant derived compounds against L. tropica parasites.In a previous work on Haplophyllum myrtifolium, the extracts and pure compounds were reported to have both in vivo and in vitro activity on L. tropica [6].The ethanol, water and n-hexane extracts from the leaves of Arbutus unedo were tested in vitro against L. tropica promastigotes and the ethanol extract of A. unedo leaves at the concentrations of 100, 250, 500 µg/ml were found to be more effective than the other extracts [33].Extracts of the fruits of M. azedarach and N. oleander brought about 97 and 81.5 % inhibition of L. tropica promastigotes respectively [34].In this study, selected Turkish medicinal plants from Eryngium, Origanum, Centurea, Phlomis and Galium genus were evaluated against L. tropica.In a previous investigation, Fokialakis et al reported that the dichlormethane extracts of Eryngium ternatum, Origanum dictamnus and Origanum microphyllum and methanol extract of Eryngium amorginum had significant in vitro anti-leishmanial activity against L. donovani [13].Antiprotozoal and antimicrobial activities of Centaureae species growing in Turkey were investigated in vitro and the highest anti-leishmanial activities among the extracts were observed with the chloroform extract of C. hierapolitana [16].
A fraction obtained from the n-hexane extract of Galium mexicanum inhibited the growth of L. donovani parasite at a concentration of 333 μg/ml for the period of 72 hours [35].The chloroform, n-hexane and water extracts of Phlomis curdica and Phlomis leucophracta were reported to possess inhibitory activity against L. donovani amastigotes [21].Thus, the present study constitutes the first report on antileishmanial activities of plant extracts of Centaurea calolepis, Phlomis lycia, Eryngium thorifolium, Origanum sipyleum and Galium incanum ssp.centrale.
The polarity and the nature of phytochemical compounds present in plant materials are the main factors that must be considered for selection of extraction solvent.The type of extractant may range from non-polar to polar solvent depending on the targeted bioactive components.In the present work, the sample preparation disparity in regarding the choice of solvent was based on the extraction yield and on our previous preliminary antiprotozoal investigations on the plant species.Water and chloroform extracts of C. calolepis, G. incanum ssp.centrale and P. lycia and the water and methanol extracts of E. thorifolium and O. sipyleum were comparatively investigated.In vitro studies have shown that all the investigated extracts exhibited 98.1-100 % inhibitory effect on L. tropica promastigotes at the highest dose of 500 µg/ml.Secondary metabolites such as alkaloids, flavonoids, saponins and terpenoids were known to possess antileishmanial activities [4,36].Thus the inhibitory effect of the extracts could be due to presence of wide range of secondary metabolites with different polarities.
In the present study, the extracts of Eryngium thorifolium and Centaureae calolepis had both in vitro and in vivo effects on L. tropica.After the 60th day of infection, live promastigotes were not detected in the groups of mice treated with E. thorifolium methanol extract.This activity was explained by the mechanism of extract either by killing parasites or causing metabolic disorders to inhibit the reproduction of parasites.
To the best of our knowledge, this is the first study of the cytotoxic effect as well as the in vivo and in vitro antileishmanial activities of these plants.

CONCLUSION
Both the in vivo and in vitro results and parasite viability findings confirm that E. thorifolium methanol extract, which possesses terpenoids, flavonoids, anthracenes and tannins, is a potential source of new and selective antileishmanial agents.

Figure 1 :
Figure 1: Cutaneous lesion on right hind mouse footpad of untreated group

Table 1 :
Medicinal plants investigated for their anti-leishmanial and cytotoxic activities

Table 2 :
Extraction yield and preliminary phytochemical analysis of plant extracts

Table 3 :
IC50 values for plant extracts tested against WI-38 cell lines