Luteolin as a potent anti-leishmanial agent against intracellular Leishmania tropica parasite

Purpose: To examine the anti-leishmanial and cytotoxic effects of five naturally occurring phenolic compounds: luteolin (1), lalioside (2), luteolin-4’-O-β-D-glucopyranoside (3), apigenin 4-O-β-Dglucopyranoside (4) and apigenin (5) on Leishmania tropica KWH23 amastigotes . Methods: The compounds were isolated from the leaves of Lawsonia Inermis via hyphenated high performance liquid chromatography-high resolution mass spectrometry coupled with solid phase extraction-tube transfer nuclear magnetic resonance technique. The isolated compounds were given intraperitoneally to L. tropica KWH23 amastigotes-infected albino mice at a dose of ≥ 3 mg/kg for 5 days. Amphotericin-B was used as standard (reference) drug. Lymphocytes were used to analyze their cytotoxicity. Results: For compound 1, mean lesion size decreased from 0.82 ± 0.12 to 0.10 ± 0.01 after 120 days, with 97 % cure of intracellular L. tropica amastigotes at a dose of 15 mg/kg, compared to amphotericin B which produced 95 % cure at a dose of 30 mg/kg. Half-maximal concentration (IC50) for compound 1 was 4.15 μg/ml against lymphocytes. Conclusion: The results indicate that luteolin is a potent inhibitor of L. tropica amastigotes, with a higher cytotoxic activity against lymphocytes, compared with luteolin-4’-O-β-D-glucopyranoside.


INTRODUCTION
Leishmaniasis, caused by parasites belonging to the genus Leishmania (Family Trypanosomatidae), is a major public health problem in tropical and sub-tropical regions.The parasite is transmitted by the sand fly (vector), with dogs, sheep, rats, horses, and cats being common animal hosts.The World Health Organization, WHO has reported that people from 98 countries in 5 continents, are at high risk of leishmaniasis, and it is estimated that approximately 12 million people are currently infected [1][2][3].Cutaneous leishmaniasis is caused by different Leishmania species, e.g.Leishmania tropica, Leishmania major, Leishmania amazonensis, and Leishmania brazillensis.In Pakistan, L. tropica and L. major are the main causes of cutaneous leishmaniasis [4,5].First line therapy for cutaneous leishmaniasis in Europe, Asia and Africa is pentavalent antimonials, i.e., sodium stibogluconate and meglumine antimoniate [2].However, antimonials have severe side effects like myalgia, pancreatitis, cardiac arrhythmia, hepatitis, and drug accumulation in liver and spleen.Thus, there is an urgent need for new chemical entities for non-toxic and effective treatment of leishmaniasis [2,4].
However, the current work deals with antileishmanial and cytotoxic effects of naturally occurring plant phenolic compounds having four flavonoids and one alkylphenone.

In vivo test
To study the pathogenesis of leishmania strain, 9 groups of male BALB/c mice (aged 6 -8 weeks, and weighing 20 -32 g) were used.Drug administration was through cardiac route. (1) (2) Promastigotes of L. Tropica KWH23 [21] were cultured in RPMI-1640 medium along with 10 % fetal bovine serum, penicillin (200 U/ml) and streptomycin (0.2 mg/ml).The parasite was cultured at 26 °C for 4 days in BOD incubator (Gallenkamp, Size 1, UK), and then harvested parasite.The harvested promastigotes were taken in a sterile tube and counted in a haemocytometer (REICHERT, N.Y, USA) under upright microscope (CX31, OLYMPUS, Tokyo, Japan).The promastigotes were centrifuged at 4 °C for 10 min at 2000 rpm; the supernatant liquid was discarded while pellet was left in tube.Fresh RPMI-1640 medium with 10 % FBS was added to get the required volume (10ml).The required volume (10 µl) of promastigotes (containing 1.4 x 10 6 promastigotes/ml) was injected into the cardiac cavity (intraperitoneally) of the BALB/c mice.Developed lesions were measured weekly with dial micrometer (Mitutoyo, Japan) during the infection period.Infection was well established and clearly visible lesions were evident to the naked eye after 36 days.Then treatment process was started.Dose of test compounds given to Groups I, II, III, IV and V were 3.0 mg/kg for 5 days (total dose = 15 mg/kg) in DMSO up to final volume of 3 ml.Amphotericin-B was used as standard drug (positive control) at a dose of 15 mg/kg.No drug agent was used in VII group (negative control).The injection dose of 10 µl was given five times with 3-day intervals, and lesion measurements were recorded regularly.Dial micrometer was used to note the difference between size of the lesion in infected and uninfected mice weekly.Before and after treatment, needle aspirations were taken from the lesions [22].To detect amastigotes under upright microscope, Giemsa stain was used, and the samples were examined under oil immersions.On the 30 th , 60 th , 90 th and 120 th day of infection, 60 mg of tissue sample was taken from the lesion for biopsy.In the identification of amastigotes, sample was smeared on the slides stained with Giemsa and upright microscope was used for examination.

Ethics statement
BALB/c mice were supplied by Department of Pharmacology (Animal center), University of Peshawar, KPK, Pakistan and this study was approved by Animal and Ethics Committee, Faculty of Pharmacy and Health Sciences, University of Balochistan (UOB), Quetta (approval ref. no.093/FOPHS/UOB).The animals were maintained in accordance with UOB, Quetta Policy and international guidelines on the care and use of laboratory animals [20].Standard diet along with water were given ad libitum to the BALB/c mice during experiments.

Cytotoxicity test
Fresh blood (10 ml) from a healthy volunteer was taken in BD vacutainer K2E (EDTA) to get mammalian cells (lymphocytes).Cytotoxic assay of test compounds was carried out by an adoption of the method described by Iqbal et al [22].PBS was passed through 0.2 µm filter under laminar flow hood (kept sterile conditions) and then equal volumes of PBS and blood were taken in a sterile tube.Ficol solution (volume ratio 1:2) was carefully added at 165° angle to the mixture of PBS and blood.The tube was centrifuged at 2000 rpm at 4 °C for 30 minutes.The lower transparent portion was punctured with a syringe, and the liquid carefully removed and added to 5 ml of RPMI-1640 medium.The number of lymphocytes was counted in a haemocytometer under upright microscope.In the next step, 100 µl of the lymphocyte media was put into each well of a 96-well culture plate.The test compounds were added at doses of 100, 50, 25 and 10 µg/ml in DMSO, each in a final volume of 3 ml.Amphotericin-B (25 µg/ml, positive control) was used as reference drug, while negative control was L. tropica KWH23 promastigotes.Using a multipipette, 10 µl of promastigotes (containing 1.4 x 10 6 promastigotes/ml) was added to 12 wells of the culture plate and placed in an incubator at 26 °C for at least 48 h.Haemocytometer was used to count viable lymphocytes and promastigotes under light microscope (lens 40x) at 24 and 48 hours.The cytotoxic tests were done in triplicate and the IC 50 for each compound was calculated [23].

Statistical analysis
Results of in vivo anti-leishmanial assay of plant extract were expressed as mean % inhibition of parasite growth ± SD (n = 3).Cytotoxicity values were expressed as 50 % Inhibitory concentration (IC 50 ) and analysed by non-linear regression analysis.For in vivo assays, mean lesion size (mm) and percentage cure were analysed by GraphPad Prism 5 software (GraphPad software, San Diego, CA) at 95 % confidence limit.

RESULTS
Results of in vivo anti-leishmanial activities of compounds 1 -5 analyzed in albino mice infected with 0.02 ml of clinically isolated L. tropica KWH23 having 1.4 x 10 6 promastigotes, via intraperitoneal route, are shown Table 1.Mean lesion size decreased significantly from 0.29 ± 0.21 mm to 0.10 ± 0.01 mm after treatment with test compounds but that of the negative group reached 1.5 ± 0.02 mm, whereas it decreased in the Amphotericin (standard drug) group from 0.85 ± 0.05 mm to 0.17 ± 0.01mm after 120 days.Luteolin showed the strongest anti-leishmanial activity.The mice groups that received apigenin, luteolin-4'-O-β-Dglucopyranoside, and apigenin 4-O-β-Dglucopyranoside had mean lesion sizes of 0.29 ± 0.21 mm, 0.25 ± 0.09 mm and 0.21 ± 0.03 mm respectively, while corresponding percentage cure were 75.02, 78.51 and 80.19 % respectively.With Luteolin and lalioside mean lesion sizes were decreased to 0.10 ± 0.01 and 0.19 ± 0.03 mm, corresponding to 97.02 and 85.27 % cure, respectively.

Cytotoxic effect
Results of cytotoxic activities of compounds 1-5 are summarized in Table 2
Although different activities pertaining to the compounds studied are available in literature, the current study is the first report on the antileishmanial activities of compounds 2 -4 and the first comprehensive in vivo study of cytotoxic activities of compounds 1-5 against L. tropica and mammalian cells.

CONCLUSION
Luteolin is a potent anti-leishmanial agent, but is cytotoxic against lymphocytes.Luteolin-4'-O-β-Dglucopyranoside possesses significant antileishmanial activity, and is least toxic against lymphocytes.Lalioside exert anti-leishmanial activity while apigenin 4-O-β-D-glucopyranoside and apigenin have a moderate inhibitory effect on intracellular amastigotes of L. tropica strain.Further studies on luteolin, luteolin-4'-O-β-Dglucopyranoside and other isolated compounds are necessary to investigate their mechanisms of action, specificity and structure-activity relationships.

Table 1 :
In vivo antileishmanial activity of plant phenolic compounds 1-5.Data represent mean lesion size (mm) with percentage cure rate with 95 % confidence intervals