Isolation of potent antileishmanial agents from Artemisia kermanensis Podlech using bioguided fractionation

Leishmaniasis is a major health problem worldwide with different clinical forms that depend on the parasite, the host's immune system, and immune-inflammatory responses. This study aimed to evaluate the secondary metabolites from Artemisia kermanensis Podlech by bioguided fractionation against Leishmania major. The chemical structures of the isolated compounds were determined based on analysis of mass and nuclear magnetic resonance spectra. Antileishmanial activity were determined on promastigotes and amastigotes. Chemical structures of the isolated compound were as 1-Acetoxy-3,7-dimethyl-7-hydroxy-octa-2E,5E-dien-4-one for compound 1 and 5,7-dihydroxy-3′,4′,6-trimethoxyflavone (Eupatilin) for compound 2, and 5,7,3′-Trihydroxy-6,4′,5′-trimethoxyflavone for compound 3. Compound 2 were confirmed by significant activity with IC50 of less than 50 μg/ml for 24 and 48 h in clinical form (amastigotes). Compound 3 demonstrated high susceptibility with an IC50 of less than 30 μg/ml for promastigotes for 24 h. The bioguided fractionation of A. kermanensis resulted the isolation of potent antileishmanial agents with a low toxicity effect on macrophages. These plant metabolites can be a candidate as a drug for treating cutaneous leishmaniasis.


Introduction
Leishmaniasis is one of the most important vector-borne diseases of humans that is caused by different species of protozoan parasites and belongs to the genus Leishmania (Attioua et al. 2011;Mesa et al. 2017;El-Khadragy et al. 2018). Among tropical infections, Leishmaniasis ranks fourth in morbidity (Monzote et al. 2014). Dogs, rodents, reptiles, and humans are the vertebrate hosts of the Leishmania parasite and the bite of a reservoir animal or human by a sandfly from the genus Phlebotomus in the eastern and Lutzomyia in the western hemisphere causes disease transmission (Kaya et al.; Monge-Maillo and López-Vélez 2013; Mesa et al. 2017). Leishmania parasite takes two forms in its life cycle: Amastigotes are round or oval shapes of the parasite with 2-4 µm length that are immobile in vertebrate host's macrophages. Promastigotes are shuttle or whip shapes with 10-20 µm length and can be diagnosed in the mosquito digestive tract (Kaya et al.). Important clinical forms of leishmaniasis include cutaneous, mucosal, and visceral forms that are different due to their immunopathology and mortality rate (Tamargo et al. 2017). The global burden of cutaneous leishmaniasis has increased, and it is endemic in 160 areas in Iran (Oliaee et al. 2020). Cutaneous leishmaniasis that caused by Leishmania tropica and Leishmania major will improve within a year but due to the scarring and subsequent problems, WHO has recommended its treatment (Kaya et al.;El-Khadragy et al. 2018). Although meglumine antimoniate (Glucantime®) and sodium stibogluconate (Pentostam®) are gold standard drugs in the treatment of leishmaniasis, but they cause serious toxicity (Aloui et al. 2016). On the other hand, alternative drugs such as amphotericin B liposomal and miltefosine for Second-line chemotherapy despite their good effectiveness, are expensive (Sharma et al. 2012;Islamuddin et al. 2015;Aloui et al. 2016). Factors such as long-term, high-dose medication regimens and serious side effects of the medications can cause leishmaniasis treatment to be stopped by patients. For these reasons, WHO has prioritized research into discovering new treatments that are more affordable, safer, more cost-effective and easier to use to improve patient's quality of life (Mesa et al. 2015(Mesa et al. , 2017Moghaddas et al. 2017). Due to this fact that in many cases of infectious and non-infectious diseases phytotherapy is used, much attention has been paid to the effects of herbal medicines and plant metabolites to make modern drugs such as anticancer and immunosuppressants and also to treat leishmaniasis (Mirzaei et al. 2016;Mesa et al. 2017).
Asteraceae family has been used since many years ago because of their expectorant, antibacterial, antifungal and antiparasitic properties (Wink 2015;Rustaiyan and Faridchehr 2021). Artemisia species from Asteraceae family have been used as a traditional medicine to treat patients with abdominal pain, uterine bleeding and inflammation. Moreover anti-coagulant, anti-diabetic and anti-tumor effects of some Artemisia species have been proved (Yun et al. 2016;Hbika et al. 2022). Terpenoids particularly sesquiterpene lactones and flavonoids are the most important secondary metabolites from Artemisia species that have been demonstrated to have antiviral, antifungal, antiprotozoal, and antitumor activities (Li et al. 2015;Wink 2015;Zamanai Taghizadeh Rabe et al. 2015). In recent years, studies have shown the inhibitory effects of ethanolic extracts of A. ciniformis, A. biennis and A. turanica against Leishmania major (Mojarrab et al. 2015).
Artemisia kermanensis Podlech named locally as Dermane Kermany is a green aromatic perennial plant that grows in central deserts and south-eastern mountains of Iran like Taftan mountain in Sistan and Baluchestan Province (Yazdiniapour et al. 2021).
Due to the importance of leishmaniasis and various drug side effects that were mentioned, in this study potent antileishmanial activity of isolated compounds from A. kermanensis has been evaluated.

Extraction and isolation
5.0 kg of all parts of A. kermanensis was air-dried and extracted by the maceration method for several times. The solvent combination was included dichloromethane and acetone (2:1). The extract after filtration was evaporated under reduced pressure to get crude extract (554.2 g). To remove the chlorophyll from the crude extract, it was coated on reverse silica gel and treated with methanol: water (7:3). After evaporating the solvent, the methanolic crude extract (288.2 g) was chromatographed by MPLC on silica gel column (15-40 µm) using a gradient solvent system from hexane 100% to ethyl acetate 100%. Three fractions were eluted with hexane/ethyl acetate: Fr 1 (20%), Fr 2 (30%), and Fr 3(40%) were subjected to evaluate their antileishmanial effect. Fractions 2 and 3 were selected for further purification according to parasitology analysis. Fraction 2, was first purified by a semipreparative HPLC direct phase column, using hexane/ethylacetate (80:20) as the mobile phase affording compound 1. Compound 2 and 3 were isolated from fraction 3 by crystallization and preparative TLC respectively. Structure elucidation of isolated compounds were obtained by extensive spectroscopic analysis, including NMR and MS.

Preparation of stock solutions
100 mg of each fraction and 10 mg of the isolated compounds were dissolved by DMSO and were obtained to 10 ml and 5 ml volume by culture medium respectively. The final concentration of DMSO was under 1%, the concentration that has no effect on proliferation of parasites or cells (Santos et al. 2008). Various concentrations (50, 100, 200, 500, 1000, 2000 μg/ml) from stock solutions of fractions were made for promastigote and amastigote assay. Concentrations include (1000, 2000, 5000, 10,000 μg/ml) of fractions were prepared to evaluate their cytotoxic effects.
Also the best concentrations of compounds that helped us to calculate IC 50 and CC 50 has been reported.
As positive control 25, 50, 100, 200 μg/ml of glucantime added to the wells for amastigote assay and amphotericin B solution with a concentration of 0.6 and 0.4 μg/ml was used for promastigote assay (Eskandarain et al. 2020).

Macrophage culture
The J774 cell line was obtained from Pasteur Institute of Tehran, Iran, cultured in cell culture flasks containing RPMI-1640 medium supplemented with %20 inactivated FBS plus 100 IU/ml penicillin and 100 μg/ml streptomycin at 37 °C and 5% CO 2 . Then the cells were harvested from the flasks using cell scrapers.

MTT assay for promastigotes
For preparing the MTT solution, 5 mg of MTT powder (BioIdea) with 1 ml sterile RPMI-1640 in dark room was combined. Logarithmic promastigotes were counted by neobar slide (3 × 10 6 /ml) and added at 100 μl into each well of the 96-well plate. Different concentrations of the fractions and pure compounds were already added. Each concentration was tested in triplicates.
After 24 and 48 h incubation at 24 ± 1 °C, 100 μl MTT solution (0.5 mg/ml) was added to each well in dark condition. The negative control was RPMI-1640 medium with parasites without treating. Also 1%DMSO plus culture medium were added into three wells as control group. Then 96-well plates were incubated at 24 ± 1 °C for 3 h and then were centrifuged at 1000 rpm for 10 min and the supernatant was drained off. DMSO (50 μl) was added to each well and absorbance was read with an ELISA reader at 570 nm. Finally, the percentage of alive promastigotes was calculated using the mentioned formula: AT: absorbance of exposed promastigotes; AC: absorbance of unexposed promastigotes; AB: absorbance of the blank.
For more reassurance, the number and morphology of the treated promastigotes were studied with neubauer chamber and direct microscopic examination.

MTT assay for macrophages
The cultured macrophages were harvested from the flasks using cell scrapers and a number of 4 × 10 4 of the cells were added to each well of the 96-well plate. Different concentrations of fractions and pure compounds were added to each well. After 24 and 48 h incubation at 37 °C and 5% CO 2 , 100 μl MTT solution (0.5 mg/ml) was added to each well and the plate was incubated at 37°c and 5% CO 2 for 3 h. Then, the supernatant was drained off of the wells and 50 μl of DMSO was added to each well. Then, absorbance was read by ELISA reader device at 570 nm. Finally, the percentage of cell viability rates were estimated using the formula that was mentioned for promastigotes.

Activity against axenic amastigote forms
The cultured macrophages were harvested from the flasks using cell scrapers and a number of 1 × 10 5 cells were transferred to each 3.5 cm well of the 6-well plates.
Prior to transferring the macrophages, the bottoms of the wells were covered with sterilized 22 × 22 mm coverslips. Stationary-phase promastigotes (1 × 10 6 ) of L. major were added to the macrophages at a ratio of (10:1). Two wells without any expose with any fraction or compound were considered as negative control.
After 24 h of plate incubation at 37 °C and 5% CO 2 , the promastigotes that did not enter the cells were removed by PBS and fresh RPMI-1640 medium was added to the wells. Then various dilutions of fractions and pure compounds were added into the wells. We picked up the slide from the Fig. 2 Percentage of promastigote viability exposed to different concentrations of fractions and pure compounds at 24 and 48 h. There is statistical difference with control group (P < 0.05). Each bar represents the mean ± SD of three independent experiments bottom of the plates 24 and 48 h later and fixed them by methanol and stained with Giemsa.
The average number of amastigotes in each macrophage was determined by studying and counting at least 100 macrophages in duplicate cultures with direct microscopic examination, to obtain the percentage of viable amastigotes.

Statistical analysis of data
Graph Pad Prism version 8.4.3. was used for statistical analyses and preparing graphs. One-Way ANOVA was applied to compare the mean's differences.

Promastigote assay
Due to the results of one-way ANOVA test and as shown in Fig. 2, Fr 2 and 3 had the best inhibitory effect on the L. major promastigotes. The IC 50 values of Fr 2 were determined 47.9 and 42.9 μg/ml and for Fr 3, 427.0 and 120.6 μg/ml at 24 and 48 h respectively. It seems that time exposure has importance in the effect of these fractions. Fr 1 did not show significant difference in comparison to control group.
According to MTT test results, the percentage of promastigote's proliferation that were treated with compound 1 that was eluted from Fr 2 by HPLC, was lower than the control group (P < 0.05) and the IC 50 values were determined 47.1 and 41.7 μg/ml at 24 and 48 h. Microscope examination tests also revealed that the alive promastigotes were morphologically deformed. We observed that exposure with high concentrations of this compound cause greater toxicity to promastigotes. Time exposure was also important, after 48 h the fatal effect has increased. As a result, the fatal effect of compound 1 for promastigotes was dose-and time-dependent. Compound 3 also with 30 μg/ml > IC 50 had antileishmanial effects against promastigotes (Table 1, Fig. 2).

Anti-amastigote assay
After removing the slides from the bottom of the 6-well plates and staining them with giemsa, it was observed that in control groups, stationary-phase promastigotes were entered the macrophages and became amastigote form.
The IC 50 values of Fr 2 were under 50 μg/ml for amastigotes at 24 and 48 h and for compound 2 were under 40 and 20 μg/ml at 24 and 48 h (Table 1).
Also, by calculating the average number of alive amastigotes that were treated with compound 1 and 2, and comparing them with the control group, it was found that these compounds have reduced the average number of amastigotes in macrophages (Figs. 3, 4).
The anti-amastigote effect of Fr 2 and 3, compound 1 and 2 was time-and dose-dependent.

Cytotoxicity assay
According to the results from MTT tests, fractions and pure compounds were not seriously toxic for macrophages (Table 1, Fig. 5). We observed that 10,000 μg/ml of the Fr2

Discussion
Cutaneous Leishmaniasis is endemic in some regions of Iran like the south, east, and central areas. First-line treatment Fig. 3 Percentage of amastigote viability exposed to different concentrations of fractions and pure compounds at 24 and 48 h. There is statistical difference with control group (P < 0.05). Each bar represents the mean ± SD of three independent experiments drugs are toxic and drug-resistant has occurred by some species of the parasite (do Monte-Neto et al. 2011). Moreover, we have distance with ready vaccines for the prevention of the disease (Read et al. 2013). Artemisinin, a sesquiterpene lactone from A. annua and its derivatives like artesunate are potent drugs for the treatment of cerebral malaria infection (Wink 2015). The effectiveness of artemisinin has been proven in cancer, obesity, allergic and auto-immune disease like ulcerative colitis (Numonov et al. 2019;Hua et al. 2022). Anti-inflammatory properties of sesquiterpene lactone from A. khorassanica was reported (Zamanai Taghizadeh Rabe et al. 2015).
Geraniol (3,7-dimethyl-2,6 octadien-1-ol) is a monoterpene alcohol with antimicrobial, antitumor and insecticidal activity. Anti-trichomonal effects of geraniol have been studied and morphological changes in the parasite has been observed (Dai et al. 2016). Compound 1 that was eluted from Fr 2 using HPLC is one of the acetylated derivatives of geraniol isolated in 1986 from A. aucheri but its biological effects had not been studied (Rustaiyan et al. 1987). Our study shown that significant reductions of promastigote viability caused by different concentrations of compound 1. For compound 1 the toxicity on macrophages is higher than 2000 μg/ml while the IC 50 value of promastigote and amastigote forms of Leishmania is less than 50 μg/ml. Eupatilin (5,7-dihydroxy-3′,4′,6-trimethoxyflavone) is one of the important lipophilic flavonoids from Artemisia species that is known for its antioxidant, anti-inflammatory, anti-apoptotic and anticancer properties and in vitro studies have shown that eupatilin could inhibit the development of atopic dermatitis symptoms in Balb/c mice that were induced by oxazolone (Li et al. 2015;Jung et al. 2018). Antileishmanial activity of 3-hydroxyflavone, luteolin, fisetin and quercetin have been demonstrated with low toxicity (Tasdemir et al. 2006). Methylated flavones (eupatilin and 5,7,3′-Trihydroxy-6,4′,5′-trimethoxyflavone) isolated from Fr 3 of A. kermanensis have showen antileishmanial activity.
Although the extracellular parasites usually result in high efficacy, eupatilin was confirmed by significant activity with IC 50 of less than 40 and 20 μg/ml for 24 and 48 h in clinical form (amastigotes). Probably eupatilin due to its lipophilic structure can inter the cells. For 5,7,3′-Trihydroxy-6,4′,5′trimethoxyflavone an extra hydroxyl group in 3′ of eupatilin converts the structure to a potent antileishmanial agent with IC 50 less than 30 μg/ml for promastigotes in 24 h. It can be predicted compound 3 has significant efficacy in the clinical form of leishmania with low toxicity according to the efficacy of the fraction source (Fr 3) of this compound.

Conclusion
In this study the bioguided fractionation of A. kermanensis was resulted to isolation of potent antileishmanial agent with low toxicity effect on macrophages. This work is the first to demonstrate the in vitro antileishmanial effect of A. kermanensis and isolated compound. With the in vivo and clinical studies, probably this plant metabolites can be a candidate as a drug for treating cutaneous leishmaniasis.
Acknowledgements This work was supported by Isfahan University of Medical Sciences, Isfahan, Iran (Grant No. 3991071) Author's contribution SS performed the experimental and wrote the manuscript draft; SS revised the manuscript, conducted the parasitological tests and analysed the data; ZY revised the manuscript and conducted the plant extraction, fractionation, purification and identification of compounds.