Anti-Leishmanial Activity of Artemisia persica, A. spicigera, and A. fragrance against Leishmania major.

BACKGROUND: Neglected tropical diseases (NTDs) like zoonotic cutaneous leishmaniasis (ZCL), is a widespread infectious disease with high mortality and morbidity. Various medications are used for treating the disease, but several side effects and drug resistance have been reported. Herbal medicines are unlimited sources for discovering new medications to treat infectious diseases. We aimed to determine the leishmanicidal activity of three species of Iranian Artemisia herbal plant extracts in in-vitro.
METHODS: In-vitro anti-leishmanial activity of ethanolic extracts on both promastigotes and amastigotes was determined by using MTT method. IC50, CC50, EC50 and SI were calculated. The study was done in 2019-2020 in Iran University of Medical Sciences, Tehran, Iran.
RESULTS: All of the three Artemisia species significantly reduced the number of parasite promastigotes. Among them, A. persica had the highest leishmanicidal activity against parasite promastigotes. Cytotoxicity assay elucidated that the Artemisia had no toxicity to the host cells, and killed the L. major amastigotes very efficiently. By increasing the dose of extracts, the parasite number in both phases (promastigotes and amastigotes) was reduced significantly.
CONCLUSION: These results indicated satisfactory anti-leishmanial activity of Artemisia extracts against ZCL in-vitro. Accordingly, Artemisia ethanolic extracts might be considered as a strong, effective and safe herbal compound for clearing the L. major with less toxicity to the host macrophages cells. Hence, it may be recognized as an excellent herbal therapy for treating the ZCL.

Introduction

Leishmaniasis is one of the most common parasitic diseases that usually produce various devastating disorders on the different parts of the body. This infection is one of the most common neglected tropical disease in the world especially in tropical and subtropical regions like Iran and has been categorized into different forms including cutaneous (CL), muco-cutaneous (MCL), and visceral (VL) types which (1–3). CL is the most prevalent form of the infection with approximately 1.5 million new cases diagnosed each year and over 370 million people are at risk of this infection throughout the world, where nearly all cases occur in several countries such as Iran, Afghanistan, Peru, Algeria, Brazil, Syria and Saudi Arabia (4, 5). Zoonotic CL (ZCL) is one of the most common forms of cutaneous lesions that usually produce various skin lesions. The causative agent of ZCL is Leishmania major, an intracellular protozoan infection, which is transmitted by a bite of sand flies to mammalian hosts.

Pentavalent antimonial compounds like glucantime and amphotericin B, are used as the first-line and second-line drugs for the treatment of CL, respectively, which have various toxic effects and lead to serious side effects such as liver, heart and biochemical disorders (6–9). Furthermore, to date, Leishmania parasites have developed resistance to existing chemical drugs (10, 11). Thus, determination and development of new medicinal agents are essential for alternative treatment. In this regards, natural herbal compounds with anti-leishmanial effects can be very useful, valuable, safe and inexpensive sources of antimicrobial agent (12, 13). Anti-leishmanial effect of some herbal plants has been attributed to presence of the materials such as quinines, terpenes, steroids and flavonoides (14, 15). In herbal medicine, one of the less-studied herbal plants in Leishmania infections is Artemisia known as “teretkh” in Iranian folk medicine which is ubiquitously present in different parts of Iran, especially in the northern part of it (16). The Artemisia genus as an aromatic perennial herb belonging to astraceae family. Among 500 species of Artemisia genus which are grown all over the world, 34 species are distributed in Iran (16, 17). Known plants for their potent chemical constituents and their essential oil which are used as a traditional medicine for the treatment of inflammatory and infectious diseases (18–23). Artemisia species have several phytochemical constituents like flavonoids, terpenoids and coumarins. In this herbal plant, various activities such as antimicrobial, antihepatotoxic, insecticides, anti-inflammatory, and antimalarials effects have been identified (16, 24–27).

To the best of our knowledge, this is the first experiment measuring the main efficacy, toxicity and antileishmanial activity of three major Iranian flora of Artemisia including A. persica, A. spicigera, and A. fragrance ethanolic extract against ZCL due to L. major in in-vitro condition and calculate the inhibitory concentration (IC50), cell cytotoxicity (CC50), effective concentration (EC50) and selectivity index (SI).

Materials and Methods

Plant material

The aerial parts of different species of Artemisia herbal plant were collected at flowering stage in October and November 2016 from Karaj city, Iran and stored in the Iranian Biological Resource Center (Essential Oil and Extract Bank). The study was done in 2019–2020 in the Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran. They identified in the herbarium of Iranian Biological Resource Center (IBRC), Tehran, Iran. After identification of three species of Artemisia including A. persica, A. spicigera and A. fragrance (plant voucher specimens: IBRC P1006575, IBRC P1000158 and IBRC P1000564, respectively), about 25 g of aerial parts (leaves) of each sample was air-dried at room temperature and grounded separately in an electric grinder and powdered using a mixer and then kept in a dark amber-colored glass bottle before extraction.

Herbal extraction

Powdered samples were macerated in ethanol (Merck) as a solvent and kept for 72 h away from light and high temperature. The ethanolic extraction processes of powdered materials of Artemisia species were applied according to the protocol as described previously (28). The extracts were sterilized by filtration by means of a membrane filter (0.22 μm). Stock solutions of this extracts were freshly prepared in 500 μl of dimethyl sulfoxide (0.2% DMSO as a solvent control) and then stored at −20 °C for evaluation of its anti-leishmanial activity. Serial dilutions of plants extracts (12.5, 25, 50, 100, 200, 400 and 800 μg/ml) and drug control (glucantime: 150 μg/ml) were prepared in RPMI1640 medium.

Parasite culture

Pathogenic Iranian strain of L. major (MRHO/IR/75/ER) promastigotes was applied in the current study. L. major amastigotes were isolated from BALB/c mice and transformed to the promastigote form (29–31). The current study does not contain any experiment with human participants or animals performed by any of the authors.

MTT assay

To determine the effects of Artemisia ethanolic extract on L. major promastigotes, amastigotes and macrophages, cell viability was determined using 3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) method (29–31). Samples optical density (OD) was measured using an ELISA plate reader (BioTek Company: USA) at 570 nm. The results were evaluated by linear regression of the inhibition percentage (32).

Promastigotes susceptibility assay (IC50)

L. major promastigotes were cultivated in the stationary growth phase in RPMI1640 at 26 °C. For the promastigotes susceptibility tests, a density of 1×106 parasite/well was counted in a neubauer chamber by a light microscope and plated in 96-well micro plates. Artemisia ethanolic extract susceptibility was determined using colorimetric MTT assay and inhibitory concentration (IC50) that caused a 50% decrease in survival of parasites was calculated (33, 34).

Macrophages culture and cell cytotoxicity assay (CC50)

J774A.1 as a mice macrophage cell line was utilized in the current experiment. Cells were cultured in RPMI-1640 medium (Sigma- Aldrich Chemicals; Germany) with heat-inactivated fetal calf serum (FCS, 10%), supplemented with penicillin and streptomycin (100 μg/ml; pH=7.4). Cell viabilities were measured using colorimetric MTT assay and cytotoxicity concentration (CC50) was determined (35).

Cell line infection and effective concentration assay (EC50)

J774.A1 macrophages (5×103 cells) were plated in 96-well culture plate in RPMI1640 medium supplemented with FCS 10%, incubated at 37 °C in CO2 5% for 24 h. Then, the stationary growth phase promastigotes of L. major were seeded into each well (1:10) and incubated at 37 °C in CO2 5% for 24 h allowing the promastigotes to penetrate and infect the cells. Later, free promastigotes were removed through washing using RPMI1640 medium. The infected macrophages were treated with several increasing concentrations of Artemisia extracts as mentioned previously for 48 h at 37 °C in CO2 5%. Finally, infected cell viability was evaluated by MTT assay and results were reported as effective concentration (EC50) that killed 50% of intracellular Leishmania parasites (36).

Selectivity Index determination

The ratio of the obtained CC50 value of the cytotoxic concentrations to the obtained EC50 value of the antileishmanial activity was determined in order to calculate the Artemisia selectivity index (SI) (37). Moreover, SI was calculated for promastigote forms of parasite (SI= CC50 Macrophages / IC50 Promastigotes) (38). At a time that the SI value is under 10, that compound has an ideal antileishmanial activity. On the other hand, the ideal Artemisia compound would be cytotoxic slowly at very high concentrations, and have antileishmanial activity at very low concentrations (higher reported values= greater Artemisia activity).

Statistical analysis

CC50, IC50, and EC50 calculations and statistical analyses were conducted using Prism 8.0 for Windows (Graphpad Prism, San Diego, CA). The differences between control and treatment groups were measured using analysis of variance (ANOVA), and differences with P-values of less than 0.05 were considered statistically significant.

Results

Effects of extracts on promastigotes and inhibitory concentration (IC50) estimation

The IC50 of A. persica, A. spicigera, and A. fragrance extracts was evaluated against promastigotes of L. major in order to estimate the IC50 values. Three studied species of Artemisia ethanolic extracts significantly affected the promastigotes growth at 48 h post incubations (Fig. 1). In this regards, the Artemisia extract inhibited the Leishmania parasite growth with the IC50 of 51 μg/ml, 200 μg/ml and 400 μl/ml for A. persica, A. spicigera and A. fragrance, respectively. Glucantime (Sanofi Aventis, France) as a positive control, which was evaluated at the concentration of 150 μg/ml, could entirely inhibit the Leishmania promastigotes growth.

Fig. 1.

The inhibitory concentration (IC50) of three species of Artemisia ethanolic extracts (increasing concentrations: 12.5, 25, 50, 100, 200, 400 and 800 μg/ml) on the Leishmania major promastigotes after 48 h by using MTT assay. All data are reported in this study as mean ± SD of three repeated experiments with same outcomes

Effects of extracts on macrophages and cell cytotoxicity (CC50) estimation

The CC50 of different concentrations of Artemisia on J774.A1 macrophages was determined at 48 h after incubations and compared with the control groups (Fig. 2). The results showed no significant differences between test groups and the control groups for each Artemisia extract (P<0.05). The CC50 was calculated to be 518 μg/ml of A. persica, 560 μg/ml of A. spicigera and 700 μg/ml of A. fragrance at 48 h after incubations, respectively (Fig. 2). According to Fig. 2, three species of Artemisia ethanolic extracts with various doses in test groups had low cytotoxic effects on uninfected macrophages at 48 hours after incubation and they had been solely toxic for cells at very high concentrations.

Fig. 2.

Cytotoxicity assay (CC50) of three species of Artemisia ethanolic extracts (increasing concentrations: 12.5, 25, 50, 100, 200, 400 and 800 μg/ml) on the macrophage cell line (J774.A1) after 48 h by using MTT assay. All data are reported in this study as mean ± SD of three repeated experiments with same outcomes

Effects of extracts on infected macrophages and effective concentrations (EC50) estimation

The EC50 of different concentrations of three Artemisia species on infected macrophages with L. major was determined at 48 h after incubations and compared with the control groups (Fig. 3). A significant reduction was seen in the percentage of infected cells after 48 h. The EC50 was calculated to be 100 μg/ml of A. persica, 100 μg/ml of A. spicigera, and 110 μg/ml of A. fragrance at 48 h after incubations, respectively (Fig. 3).

Fig. 3.

Effective concentration (EC50) values of three species of Artemisia ethanolic extracts (increasing concentrations: 12.5, 25, 50, 100, 200, 400 and 800 μg/ml) on the infected J774.A1 macrophage with Leishmania major after 48 h by using MTT assay. All data are reported in this study as mean ± SD of three repeated experiments with same outcomes

Artemisia extracts and selectivity index (SI) estimation

Three species of Artemisia extracts were active against the amastigotes of L. major with a suitable SI including 518 μg/ml, 5.6 μg/ml, 6.36 μg/ml for A. persica, A. spicigera, and A. fragrance at 48 h post infection, respectively. SI estimation elucidated that all three Artemisia extracts were actively selective against L. major promastigotes, compared to macrophages with an SI of 10.1μg/ml, 2.8 μg/ml, and 1.75 μg/ml for A. persica, A. spicigera, and A. fragrance at 48 h post infection, respectively. Hence, Artemisia extracts were strongly active against L. major promastigotes and amastigotes in comparison with alone cell line macrophages.

Discussion

Currently available drugs for treatment of leishmaniasis are pentavalent antimony derivatives which have different side effects and almost all of the anti-leishmanial chemical compounds have various obstacles to treat the infection (39). Du to no harmful effects of herbal plants on the host cells, they can be applied as potential alternatives in the development of new anti-leishmanial agents. These herbals have selective actions against parasites without reduction of host cell viability (36, 40). Therefore, one of these herbal plants is Artemisia, which can be apply as novel natural compounds. Several studies have been proposed that Artemisia spp. may have biological functions for various clinical applications (23, 41–43).

This is the first report on comparison of in–vitro anti-leishmanial activity of Iranian Flora A. persica, A. spicigera, and A. fragrance ethanolic extract and against L. major in cell culture model. In the current study, we calculated IC50, CC50, and EC50 of three species of Artemisia extracts in order to specify their anti-leishmanial activities. The achieved data exhibited that studied Artemisia extracts have dose-dependent anti-leishmanial effects and had the inhibitory functions on the growth of L. major. In addition, these herbal plants are identified as anti-leishmanial agent against both form of parasite including extracellular promastigotes and intracellular amastigotes, while they have no harmful side effects for the host macrophages. The finding of the current study illustrated no toxicity in mice macrophages with even high dose of Artemisia extracts, which confirms its lowest harmful effects. According to published reports, other species of Artemisia such A. campestris, A. herba-alba Asso and A. aucheri plants were promising candidates as antileishmanial products which proved to be effective compounds against viability and proliferation of both promastigotes and amastigotes of Leishmania (44, 45). Their findings support our results. Furthermore, the antileishmanial activities of other Artemisia species were also described. A. absinthium from Cuba was able to clear L. amazonensis promastigotes with 50% inhibitory concentration (46). A. annua, was also potent against L. donovani (47). A. absinthium from Ethiopia has been documented to exhibit activity against the promastigote of both L. aethiopica and L. donovani strains forms (48). All the mentioned experiments are in line with the current findings.

Among these three ethanolic Artemisia extracts, A .persica showed the most potent leishmanicidal activity (IC50: 51 μg/ml) while, A. fragrance extract had less activity against L. major promastigotes (IC50: 400 μg/ml) after 48 h. The finding are in agreement with other studies. For instance, other Artemisia species such as A. kulbadica and A. ciniformis revealed the good antileishmanial effects (49). An in vitro study similar to our study was conducted to examine the effect of A. absinthium extract on the growth of L. major promastigotes. The results indicated that after 48 h of exposure, the IC50 of A. absinthium extract was 56 mg/ml and the leishmanicidal effect of concentrations greater than 200 mg/ml almost 80% (50). The results indicated that the optimal concentrations of the A. spicigera and A. persica extracts for reducing the amastigotes growth inside the macrophages were 100μg/ml that killed more than half of amastigotes forms. The current study has indicated that the SI values for three Artemisia extracts were approximately under 10, that these compounds had ideal anti-leishmanial activity and toxic for L. major promastigotes and amastigotes forms, in comparison with the host cell.

The leishmanicidal activities of various herbal plants such as artemisinin, racemoside, curcumin, Piper betle and Aloe vera have been documented to be interceded by apoptosis (47). Leishmania as an intracellular parasite can undergo programmed cell death in response to herbal natural products (47, 51). We found that, Artemisia spp. had effective leishmanicidal activity as evidenced by shrinkage in the Leishmania promastigotes cell wall that became round in shape and had no motility with disrupted flagella, which has also been seen in the previous study (47).

Conclusion

We reported antileishmanial activities of three species of Artemisia plants including A. persica, A. spicigera, and A. fragrance, which are growing in Iran. All of them exhibited potent inhibitory and antiparasitic activity against L. major promastigotes and amastigotes. This is the first experiment to our knowledge that describes strong antileishmanial activity of Artemisia against L. major. Further studies are needed to identify the various bioactive compound(s) of mentioned Artemisia species with a view to investigate them as potential alternative herbal plants to the available drugs for treatment leishmaniasis.