Ex vivo study of Anti-leishmanial activity of Artemisinin against

Leishmania tropica amastigote

Ghuffran Muhammed Hassan

Department of Biology, College of Science, University of Baghdad, Al-Jaderyia Campus, Baghdad, Iraq

*Corresponding Author E-mail: guffran_bio@yahoo.com

ABSTRACT:

Leishmania parasites are the causative agent of leishmaniasis. Many studies are inspecting chemical drugs, including the use of miltefosine and amphotericin B, but curative values may be limited for these drugs with side effects due to the chemical origin, therefore, investigating less toxic therapies is essential. The aim of this study was to investigate the effectiveness of artemisinin on Iraqi strain of Leishmania tropica, by experimental macrophage ex vivo infection of amastigotes into mouse macrophage cell-line RAW264.7. Different concentrations (100, 200, 300, 400, 500)µM of artemisinin (ART) were screened to examine the susceptibility of

L. tropica amastigotes to invade macrophage cell line along three times of follow up (24, 48 and 72) hours. Results showed that artemisinin had a cytotoxic effect on the parasite in which a significance difference (P ≤ 0.05) in cell viability was observed and IC50 was calculated as 182.6 µM after 48 hours treatment. In addition, percentage of infectivity of intracellular amastigotes was significantly decreased. These findings revealed the potential efficacy of artemisinin against the infectious amastigotes and can be further studied to screen its effectiveness in vivo for exploring a safer herbal compound to treat cutaneous leishmaniasis.

KEYWORDS: Artemisinin, cutaneous leishmaniasis, RAW264.7, amastigotes.

INTRODUCTION:

Leishmaniasis is an eukaryotic parasitic infection which is endemic in 98 countries, with more than 300 million people at threat globally and 0.7–1.3 million new cases each year. Sandflies transmit the infection to humans by inoculating Leishmania meta-cyclic promastigotes into the exposed skin, resulting in Cutaneous, Mucocutaneous, or Visceral leishmaniasis^1,2. Leishmaniasis (Visceral and Cutaneous) is one of the neglected tropical diseases in Iraq³.

The first step to control neglected tropical diseases including leishmaniasis is the development of medications⁴. Available therapies regimens for leishmaniasis have several after effect, such as high cost, high toxicity, development of resistance, relapse and poor compliance due to painful injections for long durations and sometimes a scarcity of medicines⁵.

Major medical therapies for leishmaniasis, involving sodium stibogluconate (an antimony medication) and amphotericin B (an antifungal medication), are expensive and have unwanted side effects such as abdominal pain, nausea, and anorexia, leading to huge numbers of the infected populations remaining unresponsive. Therefore, many plants have been considered for the therapy of parasitic diseases⁶. Such as Artemisinin and its derivatives (collectively termed as Artemisinins) are sesquiterpene lactones derived from the sweet wormwood (Artemisiaannua), which was identified in Chinese traditional medicine for thousands of years as a cure for fevers and chills⁷.

Artemisinin is relatively facilely purified after extraction from plants⁸. Following their discovery and development of antimalarial drugs by You you Tu group in the 1970s⁷. Artemisinin and its derivatives have been investigated in treating parasitic diseases or parasitic infections caused by protozoan parasites including Leishmania spp., Toxoplasmagondii, Trypanosoma spp., Neosporacaninum, Naegleriafowleri, Eimeriatenella, Cryptosporidiumparvum, Acanthamoebacastellanii, Giardialamblia, and Babesia spp.⁹. They are potential in inhibiting the parasite metabolism while showing limited

effects on the host, indicating a higher safety index of the drugs. A large number of in-vitro or ex-vivo screening have shown that ART and its derivatives have activities in controlling the parasites, and the herbal compound shown effective against the protozoan¹⁰. In this study, artemisinin efficacy was screened against the infectious intracellular amastigotes of L. tropica inside macrophage host (RAW264.7) where cell viability and infectivity index were demonstrated by fluorescent microscopy.

MATERIAL AND METHODS:

Artemisinin (ART) preparation:

In brief, 3 mg of artemisinin was dissolved in 500 ml of Dimethyl sulfoxide (DMSO). From the stock solution, different concentrations of ART was prepared as following (500, 400, 300, 200, 100)μM.

Leishmania tropica promastigotes culture:

Department of Biology, College of Science, University of Baghdad provided Leishmania tropica promastigote isolate which was previously diagnosed by PCR in 2016¹¹. Procyclic promastigotes of L. tropicawere cultured in M199 medium (Sigma Aldrich St. Louis, MO, USA). The medium was prepared according to manufacturer’s procedure at pH 7.4 supplemented with 10% heat-inactivated fetal calf serum (HIFCS), 100 IU/ml of penicillin and 100μg/ml of streptomycin, culture was incubated at 26°C for 48-72 hours to allow proliferation of promastigotes into log phase¹².

In vitro amastigotes differentiation:

Axenic metacyclogenesis was induced by transferring the procyclic promastigotes grown at 26ºC, 10% HIFBS, cultured in M199 medium, pH 7.2 and 1% of Penicillin (50u/ml) Streptomycin (50 μg/ml) solution, the promastigotes were seeded at 5 ×10⁶ parasite/ml. The production of axenic amastigotes was induced by transferring the procyclic promastigotes of to a new RPMI140 medium supplemented with 20% HIFBS, pH 5.5, and incubated at 35°C for two 48 hours to produce the metacyclic infectious amastigotes¹³.

Macrophage host RAW264.7 cell line:

RAW264.7 the cell line was cultured at 37ºC under 5% CO2- humidified incubator in Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% HIFBS and 1% penicillin/streptomycin.

Macrophage cytotoxicity (Trypan Blue assay):

This test was used to examine the minimum concentration of ART that may be cytotoxic on RAW264.7 macrophages cell line. The macrophages were cultured on 6 well plate (Nunc^®) at a concentration of 1 x 10⁵ cells/ml as required to be added to each well in DMEM medium (SANTA CRUZ) supplemented with

10% HIFBS. The macrophages were treated with the concentrations of (500, 400, 300, 200, 100)μM of ART.

Plates were incubated at 37˚C in 5% CO2 for 24, 48, 72 hours¹⁴. Cells were stained with trypan blue and were examined by direct counting under light microscope.

Ex-vivo infection of RAW264.7 macrophages with L. tropica amastigotes:

ART was added to RAW264.7 plates with the following concentrations (500, 400, 300, 200, 100)μM. Amastigotes were suspended in DMEM and infection of macrophages was made by inoculating each well with amastigotes suspension in a ration of 1:10. Plates were prepared in triplicates and incubated for 24 hours at 37°C in 5% CO2 incubator to allow maximum internalization of the amastigote forms into macrophage cells^15,16. Control plates were treated as earlier but DMSO was added instead of ART.

Nucleic acid staining and Fluorescent microscopy: DAPI stain (4′,6-diamidino-2-phenylindole) powder was prepared according to Santa Cruz manufacture in which 2 mg dissolved in 200μl of DMSO. The stain was diluted 40 X in PBS and was added to each well for 5 minutes, then washed three times in PBS, triplicates were prepared for each concentration¹⁷.

The plates examined under fluorescence microscope. Random fields were examined to count at least 100 macrophages in each well to calculate the number of macrophages that are infected or uninfected and the total number of amastigotes within the infected macrophages, in order to measure the percentage of infection and proliferation of parasites within the macrophage host, with or without ART treatment.

Statistical analysis:

Prism Graph v.7 was used for significance estimation (p value ≤ 0.05).

RESULTS AND DISCUSSION:

Macrophage cytotoxicity (TB assay):

The results demonstrated that Artemisinin had low cytotoxicity on normal healthy culture of RAW264.7 cell line (figure-1); no more than 15% of cells was affected in which a minimum cell viability was 86% along the different concentrations used and follow-up. Trypan blue assay is an extremely common to estimate cytotoxicity in the experimental search. This method is based on the cell membrane permeability¹⁸. Similar previous study approved the low toxicity of ART on BALB/c macrophages¹³. Another research proved that ART is effective in inhibiting the parasite metabolism while showing limited antagonistic effects on the host, indicating a higher safety index of the of compound^19,20.

Figure -1: Cytotoxicity of RAW264.7 cell line with ART, cell line stained with trypan blue, picture was taken under light microscope (40 X).

Cell viability and infectivity index of amastigote infection in RAW264.7:

Artemisinin is a secondary metabolite of the herb Artemisia annua, which had proved a major potential in the therapy of experimental of many anti-parasitic²¹. Screening of ART in experimental intra-cellular amastigote-macrophage infection is one of the very first trials on the Iraqi strain of L. tropica (figure-2). The RAW264.7 mouse macrophage cell line is often utilized to the initial screen natural products for bioactivity and to investigate the possibility to affect in vivo on primary cell culture²². Furthermore, several studies investigated the toxicity of artemisinin and its derivatives such as artemether on different mammalian cells and the results exhibited low cytotoxicity, In contrary, amphotericin B was cytotoxic to human macrophage cell line U-937 in which LC50 was 30 µg/mL²³.

Figure-2: The growth of RAW264.7 cell line, the picture was taken under inverted microscope (40 X)

Cell viability was measured by plotting the number of infectious amastigotes in the host cell (100 macrophages) against ART concentrations (figure-3), maximum inhibition was observed at the high concentration of 500μM. The IC50 was calculated after 48 hours treatment and was equal to 182.6μM. Similar study showed that artemisinin inhibited the in vitro growth of L. major promastigotes, its inhibitory activity toward promastigotes determined the IC50 values of 283 μM²⁴. While in another study on L. donovani, the IC50 of artemisinin was 160μM for promastigotes, which more reduced to 22μM in amastigotes²⁵. Similar experiments proved an important decline in the proliferation of amastigotes inside the lesion's macrophages in the artemisinin treated mice compared with the other macrophages when exposed to microorganisms, they demonstrated a blast in oxygen consumption coincident with the production of nitric oxide in ART-treated cells²⁶. Moreover, Artemisinin derivative also found to have an apoptotic effect on both amastigotes and promastigotes, in vitro²⁷.

Figure-3: Cell viability of intracellular amastigotes against Artemisinin concentrations.

Infectivity index demonstrated the inhibitory efficacy of artemisinin against the infectious amastigotes to invade macrophages in ART-treated cells in comparison to ART-untreated cells (figure -4) after 24, 48 and 72 hours of follow up. Ex-vivo infection after 48 hours (table-2) showed a significant difference between test and control at all concentrations studies in which a minimum invasion percentage was 22% in 500μM compared with control plate, which was 80%. While after 24 and 72 hours, significant infectivity decrease was observed at concentrations of 200-500μM (table1, 3). The number of virulent amastigotes was calculated inside 100 macrophages and the highest significance decrease was after 48 hours incubation at all concentrations, while after 48 and 72 hours, significant decrease was noted at concentrations 200-500μM.

Figure -4:Ex-vivo infection of RAW264.7 with L. tropica amastigotes, nuclei of parasite and host cell stained with DAPI, fluorescent microscope images (40 X).

Table -1: Infectivity index of RAW264.7 with L. tropica amastigotes counted after 24 ART-treatment under fluorescent microscope.

Parameter	500 µM	400 µM	300 µM	200 µM	100 µM	Control
Percentage of infected MØ	22%	30%	40%	40%	60%	75%
Percentage of non- infected MØ	78%	70%	60%	60%	40%	24%
# Of amastigote/100 macrophages	55	40	76	70	78	80
Percentage of amastigote /cell	2.5	1.3	1.9	1.8	1.3	1.1

Table -2: Infectivity index of RAW264.7 with L. tropica amastigotes counted after 48 ART-treatment under fluorescent microscope.

Parameter	500 µM	400 µM	300 µM	200 µM	100 µM	Control
Percentage of infected MØ	20 %	24 %	25 %	30 %	40 %	80 %
Percentage of non- infected MØ	80 %	76 %	75 %	70 %	60 %	20 %
# Of amastigote/100 macrophages	26	20	22	36	50	85
Percentage of amastigote /cell	2.5	1.3	1.9	1.8	1.3	1.1

Table -3: Infectivity index of RAW264.7 with L. tropica amastigotes counted after 72 ART-treatment under fluorescent microscope.

Parameter	500 µM	400 µM	300 µM	200 µM	100 µM	Control
Percentage of infected MØ	40 %	50 %	55 %	70 %	80 %	90 %
Percentage of non- infected MØ	60 %	50 %	45 %	30 %	20 %	10 %
# Of amastigote/100 macrophages	44	60	70	78	84	90
Percentage of amastigote /cell	1.1	1.2	1.3	1.1	1.1	1

Surprisingly, the percentage of amastigote per cell (parasite load) was higher in treated cells compared with untreated cells after 24 and 48 hours of follow up, this may indicate that the low number of amastigotes, which was survived inside the macrophage, was still alive. Such results revealed dose-dependent anti-leishmanial activity of artemisinin, the longer incubation of ART lead to irreversible stress damage; this property may explain the renewed parasitic activity or recrudescence in relative short-term usages of ART²⁸. However, similar study on Leishmania tropica promastigotes in vitro showed that growth inhibition reduced the number of promastigotes in culture²⁹.

Leishmania donovani effectors that can support macrophage infection; infected THP-1 cell-line stained with DAPI, marked both the macrophages and Leishmania nuclei³⁰. This allowed the number of infected cells per total macrophages (infection rate) and the number of Leishmania per infected macrophage (parasite load) to be counted on each image and the averages were calculated as well. Comparable data published by³¹ Sen et al. (2010) showed that artemisinin effect on intracellular Leishmania-infected BALB/c mice eliminated the intra-cellular amastigotes by the generation of iron–Artemisinin compounds that observed in experimental visceral leishmaniasis. Furthermore, iron is an essential co-factor for chemical and biological pathways, including replication of all organisms including pathogens occupying within macrophages^32,33.

Artemisinin and miltefosine drugs have been reported to produce free radicals inside the Leishmania species, which possibly participate in its anti-leishmanicidal activity³⁴. Growing herbal artemisinin and its derivatives are utilized worldwide in the traditional therapy of several diseases, it provides a new class of quite effective anti-malarial due to the presence of an endo- peroxide sesquiterpene lactone³⁵.

Artemisinin action in reducing the activation of host macrophages, decreasing the production of fatal nitric oxide (NO) and restoring normal NO production in L. major-infected macrophages to elicit programmed cell death³⁶. In addition, Artemisinin facilitates

externalization of phosphatidylserine and leads to the lose of mitochondrial membrane activity, cell-cycle detention at the sub-G0/G1 phase, and apoptosis of L. donovaniextracellularpromastigotes¹⁹. Furthermore, surplus iron binds to hemoglobin and stimulates Artemisinin by assisting the formation of intra parasitic heme-iron, which catalyzes the cleavage of the endo- peroxide ring and enables the transfer of an oxygen atom from the peroxide group to a chelated iron ion, generating Fe (IV) O species. As a result, free radical intermediate or iron-ART adduct then effectively kill the promastigotes of Leishmania spp. by alkylation³¹.

CONCLUSION:

Artemisinin reduced the infectivity of infectious intracellular amastigotes of cutaneous Leishmania tropica in its intrinsic mammalian host of macrophages and may be presented as a novel remedy against Leishmania infection. Further studies of this compound are recommended for in vivo investigation on cutaneous and visceral form of Leishmania parasite.

REFERENCES:

1. Showler AJ, Boggild AK. Cutaneous leishmaniasis in travellers: a focus on epidemiology and treatment in 2015.Curr Infect Dis Rp. 2015; 17(7):37.

2. Subathra DC, Harini S, Renuka EJ, et al. Leishmaniasis: A review on global epidemiological trends. Res J Pharm Tech. 2014; 7(5): 569-574.

3. Salam N, Al-Shaqha WM, Azzi A. Leishmaniasis in the Middle East: incidence and epidemiology. PLoS Negl Trop Dis. 2014; 8(10): e3208.

4. Alvar, J Yactayo S and Bern C. Leishmaniasis and poverty. Trends Parasitol.2006; 22 (12): 552-557.

5. Den Boer M Davidson RN. Treatment options for visceral leishmaniasis. Expert Rev Anti Infect Ther. 2006; 4(2):187-97.

6. Moghaddas E, Khamesipour A, Mohebali M, Fata A. Iranian Native Plants on Treatment of Cutaneous Leishmaniosis: A Narrative Review. Iran J Parasitol. 2017; 12(3):312.

7. Wong YK, Xu C, Kalesh KA, et al. Artemisinin as an anticancer drug: recent advances in target profiling and mechanisms of action. Med Res Rev. 2017; 37(6):1492-517.

8. Heydari FE, Ghaffarifar F, Soflaei S, et al. Comparison between in vitro effects of aqueous extract of Artemisia seiberi and artemisinin on Leishmania major. Jundishapur J Nat Pharm Prod. 2013; 8 (2): 70.

9. Loo CS,Lam NS, Yu D, et al. Artemisinin and its derivatives in treating protozoan infections beyond malaria. Pharmacol Res.2017; 117:192-217.

10. Berens RL, Brun R, Krassner SM. A simple monophasic medium for axenic culture of hemoflagellates. J Parasito. 1976; 62(3):360-5.

11. Kamil MM, Ali HZ. Using PCR for detection of cutaneous leishmaniasis in Baghdad. 2016; IJS.57(2B):1125-30.

12. Efferth T. Cancer combination therapies with artemisinin-type drugs. Biochem Pharmaco. 2017;139:56-70.

13. Habibi P, Sadjjadi S, Owji M,et al. Characterization of in vitro cultivated amastigote like of Leishmania major: a substitution for in vivo studies.2008; IJP.6-15.

14. Ghaffarifar F, Heydari FE, Dalimi A, et al. Evaluation of apoptotic and antileishmanial activities of Artemisinin on promastigotes and BALB/C mice infected with Leishmania major. Iran J Parasitol. 2015; 10(2):258.

15. Rouzer CA, Jacobs AT, Nirodi CS, et al. RAW264.7 cells lack prostaglandin-dependent autoregulation of tumor necrosis factor-α secretion. J Lipid Res. 2005; 46(5):1027-37.

16. Li R-J, Gao CY, Guo C, et al. The Anti-inflammatory Activities of Two Major Withanolides from Physalis minima Via Acting on NF-κB, STAT3, and HO-1 in LPS-Stimulated RAW264.7 Cells Inflammation. 2017; 40(2):401-13.

17. Pimpinelli S, Bonaccorsi S, Fanti L, et al. Preparation and analysis of Drosophila mitotic chromosomes. 2000; Drosophila protocols.3-23.

18. Zanatta G, Steffens D, Braghirolli DI, et al. Viability of mesenchymal stem cells during electrospinning. Braz J Med Biol Res. 2012; 45(2):125-30.

19. Sen R, Bandyopadhyay S, Dutta A, et al. Artemisinin triggers induction of cell-cycle arrest and apoptosis in Leishmania donovani promastigotes. J Med Microbiol. 2007; 56(9):1213-8.

20. Want MY, Islamuddin M, Chouhan G, et al. Therapeutic efficacy of artemisinin-loaded nanoparticles in experimental visceral leishmaniasis. Colloids and Surfaces B: Biointerfaces. 2015; 130:215- 21.

21. Wink M. Medicinal plants: a source of anti-parasitic secondary metabolites. Molecules. 2012; 17(11):12771-91.

22. Merly L, Smith SL. Murine RAW 264.7 cell line as an immune target: are we missing something? Immunopharmacology and Immunotoxicology. 2017; 39(2):55-8.

23. Mesa LE, Vasquez D, Lutgen P, et al. In vitro and in vivo antileishmanial activity of Artemisia annua L. leaf powder and its potential usefulness in the treatment of uncomplicated cutaneous leishmaniasis in humans. Rev Soc Bras Med Trop. 2017; 50(1):52-60.

24. Haniloo A, Nemati S, Nahrevanian H, et al. In Vivo and In Vitro Investigation on Anti-Leishmanial Efficacy of Artemisinin on Iranian Strain of Leishmania major. Adv Stud Med Sci. 2016; 4(1):1-11.

25. Ganguly S, Bandyopadhyay S, Bera A, et al. Antipromastigote activity of an ethanolic extract of leaves of Artemisia indica. Indian J Pharmacol. 2006; 38(1):64-5.

26. Erel O, Kocyigit A, Bulut V, et al. Reactive nitrogen and oxygen intermediates in patients with cutaneous leishmaniasis. Mem Ins Oswaldo Cruz. 1999; 94(2):179-83.

27. Ebrahimisadr P, Ghaffarifar F, Hassan ZM. In-vitro evaluation of antileishmanial activity and toxicity of artemether with focus on its apoptotic effect. IJPR. 2013; 12(4):903.

28. Straimer J, Gnädig NF, Witkowski B, et al. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates. Science. 2015; 347(6220):428-31.

29. Hassan GM, Ali HZ. In-vitro effects of the Artemisinin on L. tropica promastigotes. (IOSR-JPBS). 2017; 12 (6): 82-6.

30. Colineau L, Clos J, Moon KM. Leishmania donovani chaperonin 10 regulates parasite internalization and intracellular survival in human macrophages. Med Microbiol Immunol. 2017; 206(3):235-57.

31. Sen R, Ganguly S, Saha P, et al. Efficacy of artemisinin in experimental visceral leishmaniasis. Int J Antimicrob Agents. 2010; 36(1):43-9.

32. Marquis J-F Gros P. Intracellular Leishmania: your iron or mine? Trends Microbiol. 2007; 15(3):93-5.

33. Das NK, Biswas S, Solanki S, et al. Leishmania donovani depletes labile iron pool to exploit iron uptake capacity of macrophage for its intracellular growth. Cell Microbiol. 2009; 11(1):83-94.

34. Rahaman M, Ghosh S, Chowdhury LD, et al. Evaluation of anti- leishmanial activity of artemisinin combined with amphotericin B or miltefosine in Leishmania donovani promastigotes. IJBCP. 2014; 3(4): 125-132.

35. Bora KS, Sharma A. The genus Artemisia: a comprehensive review. Pha Biol. 2011; 49(1):101-9.

36. Nemati S, Nahrevanian H, Haniloo A, et al. Investigation on nitric oxide and C-reactive protein involvement in antileishmanial effects of artemisinin and glucantim on cutaneous leishmaniasis. Adv Studies Biol. 2013; 5:27-36.

Received on 21.10.2019 Modified on 28.11.2019

Research J. Pharm. and Tech. 2020; 13(8):3787-3791.

DOI: 10.5958/0974-360X.2020.00670.8