In vitro Evaluation of the Potent Antileishmanial Activity of Ferula tabasensis Alone or in Combination with Shark Cartilage Extract Against the Standard Iranian Strain of Leishmania major (MRHO/IR/75/ER)

Background The available drugs for the treatment of leishmaniasis are highly toxic and extremely expensive, with low efficiency; therefore, the development of effective therapeutic compounds is essential. Objectives The present study aimed to explore the antileishmanial effects of ethyl acetate extract, methanol extract, and fractions 1-4 (F1-F4) of Ferula tabasensis, alone or in combination with shark cartilage extract (ShCE), on L. major in vitro. Methods In this study, ethyl acetate, methanol, and n-hexane extracts were extracted from the aerial roots of F. tabasensis by the maceration method. The silica gel column chromatography was used to separate n-hexane extracts at varying polarities (F1-F4 fractions). Subsequently, the effects of extracts and fractions against promastigotes were assessed by the parasite counting method microscopic inhibition test and MTT assay. Besides, their effects on the infected macrophage cells and the number of amastigotes were investigated. Cytotoxicity was evaluated in non-infected J774A.1 macrophage cells. Finally, apoptosis induction of promastigotes, including infected and non-infected macrophages, was evaluated. Results The results indicated the highly potent activity of F. tabasensis extracts and F1-F4 fractions, alone or in combination with ShCE, against L. major promastigotes and amastigotes in a dose-dependent manner (P < 0.05). The F1 fraction and methanol extract showed markedly higher toxicity compared to the other extracts and fractions, with 50% inhibitory concentrations (IC50/72h) of 2.4 ± 0.29 and 2.9 ± 0.55 µg/mL against promastigotes and 1.79 ± 0.27 µg/mL and 1.39 ± 0.27 µg/mL against amastigotes (P < 0.001). Moreover, they had a high selectivity index (SI) due to the low toxicity of macrophages (P < 0.0001). The results of flow cytometry indicated that the percentages of apoptotic promastigote cells in contact with IC50 concentrations of F1 and methanol extract alone after 72 h were 43.83 and 43.93%, as well as 78.4%, and 65.45% for their combination with ShCE, respectively. Also, apoptosis of infected macrophages induced by F1 and methanol extracts was estimated at 68.5% and 83.7%, respectively. Conclusions In this study, the F1 fraction and methanol extract of F. tabasensis showed potent efficacy against L. major, associated with low toxicity and apoptosis induction. Therefore, they can be promising therapeutic candidates in future animal and even human studies.


Background
Leishmania species, as obligate intracellular flagellate parasites, are responsible for leishmaniasis, which is an infectious disease affecting humans and animals (1).Leishmaniasis is recognized as an important vector-borne zoonotic disease, which commonly manifests as visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis, and diffuse cutaneous leishmaniasis (2).Nevertheless, limited effort has been made to control and prevent CL due to challenges, such as vector or main reservoir control.Among these challenges, the treatment of CL is the most important one (3).Different types of synthetic drugs are used for CL treatment, the majority of which have side effects and unsatisfactory therapeutic effects (4).Therefore, researchers are seeking more effective drugs to replace the current ones for the treatment of CL.Several natural compounds, including alkaloids, naphthoquinones, neolignans, chalcones, triterpenoids, and lignans, have been reported to exert inhibitory effects against Leishmania species (5).

Objectives
The present study mainly aimed to evaluate the leishmanicidal activity, cytotoxicity, and apoptotic affinity of ethyl acetate extract, methanol extract, and F1-F4 fractions of F. tabasensis, used alone or in combination with ShCE against L. major in vitro.

Plant Samples
The roots of F. tabasensis were collected from Tehran, Iran, in July 2020.A voucher specimen (ARD-R1) was deposited in the herbarium of the School of Pharmacy of Ardabil University of Medical Sciences, Ardabil, Iran.

Isolation of Extracts and Fractions
The aerial roots of F. tabasensis (1.5 kg) were powdered and extracted with n-hexane (2×10 L), ethyl acetate (2 × 10 L), and methanol (2×10 L) by the maceration method at room temperature, respectively.The n-hexane extract was concentrated in a rotary evaporator to obtain 45 g of a dark sticky residue.The residue was then fractionated by silica gel column chromatography (230-400 mesh size, 400 g) with an n-hexane/EtOAc gradient (100:0 to 0:100) as eluent, followed by increasing concentrations of methanol (up to 20%) in ethyl acetate.Finally, F1-F4 fractions with different polarities were obtained (22).The extracts and fractions were dissolved in 1% methanol to prepare a stock solution.It is worth mentioning that previous studies have attributed no toxicity to methanol concentrations up to 1% against promastigotes or amastigotes (23).

Cultivation of L. major
The standard Iranian strain of L. major (MRHO/IR/75/ER) was prepared by Dr. Hajjaran from the School of Public Health of Tehran University of Medical Sciences (Tehran, Iran).The promastigotes were cultured and sub-cultured in the NNN and RPMI-1640 media, which were respectively enriched with 20% fetal bovine serum (FBS) inactivated at 56ºC for 30 minutes and 1% penicillin/streptomycin (Pen-Strep) at a temperature of 24°C ± 2.

J774A.1 Macrophage Cell Culture
The murine macrophage cell line, J774A.1, which was purchased from the Pasteur Institute of Iran, was cultured in RPMI-1640 medium, supplemented with 12% FBS and 1% Pen-Strep at 37°C under humidified conditions in a 5% CO 2 atmosphere.The confluent cells were obtained by adding fresh media to the flasks daily (21,24).

Preparation of ShCE
The purchased shark cartilage (Bushehr Port, Persian Gulf, Iran) was used to prepare ShCE, according to the method proposed by Hassan et al., as described in our previous studies.In brief, 10 g of cartilage powder (the cartilage was cleaned, cut into small pieces, lyophilized, and powdered) was poured into 100 mL of phosphate-buffered saline (0.1 M PBS) containing guanidine hydrochloride (4 M) and phenylmethylsulfonyl fluoride (1 mM PMSF, pH=5.8) as a protease inhibitor and incubated at 2 -8°C for 48 hours on slight stirring.Subsequently, the solution was centrifuged at 100,000 g for 45 minutes.The supernatant was then precipitated in 20% polyethylene glycol and dialyzed against PBS.The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method was also used to 2 Iran J Pharm Res.2023; 22(1):e136173.
evaluate the purity of proteins and represent their molecular weight against the standard protein ladder (19,25).Amphotericin B (AmB, Gilead UK), prepared in sterile PBS based on the manufacturer's instructions right before the experiments, and glucantime (GLU, 1.5 g/5 mL ampules; Sanofi-Aventis, France) were used as positive controls at different concentrations (1.56 -200 µg/mL).The three wells of each plate without any drugs were considered as the negative controls (Ctrl-).The experiments were performed in triplicate (19,26).
After 72 hours of incubation at 25 ± 1°C, the plates were centrifuged at 3000 rpm for 10 minutes, and the supernatant was removed and replaced with a fresh medium of the same volume.Next, 20 µL of MTT solution (5 mg MTT powder/mL in PBS) was added to each well and incubated for five hours at 37°C in a dark room.Next, the cells were centrifuged again at 3000 rpm for 10 minutes, and 100 µL of dimethylsulfoxide (DMSO) was added to the pellets.After 10 minutes, the optical densities (ODs) were measured at a wavelength of 570 nm using an ELISA reader.The viability percentage of promastigotes was determined based on the following formula: Viability percentage = 100 × (Absorbance of treated cells -Absorbance of the blank/Absorbance of control cells -Absorbance of the blank) Meanwhile, the promastigotes suspended in PBS with no drugs and medium with no promastigotes and drugs were respectively used as the negative control (Ctrl-) and blank (19,27,28).

Intracellular Amastigote Assay
The effects of F. tabasensis extracts and fractions on infected macrophage cells and intra-amastigotes were evaluated, as previously described (19).Briefly, 3×10 5 of J774A.1 macrophage (200 µL) was added to each well of 12-well plates, with small round glass coverslips at the bottom of the plate, and incubated for 12 hours at 37°C in a 5% CO 2 atmosphere for adherence.Next, the suspended macrophages were removed by washing them with sterile PBS.To infect the macrophages, 3 × 10 6 stationary-phase promastigotes of L. major (1:10 ratio) were added to the adherent macrophages and incubated again for 12 hours at 37°C in a 5% CO 2 atmosphere with 95% relative humidity.
Free parasites were removed by washing with fresh RPMI-1640 medium after 12 hours.The infected macrophages were incubated in the presence of the extracts and fractions at different concentrations (1.56 -200 µg/mL).After 72 hours, the slides were washed with PBS, fixed with methanol, and stained with Giemsa stain to count the infected macrophages and amastigotes under an optical microscope.The GLU and AmB were used as the positive controls at the same concentrations described in the promastigote assay.The number of amastigotes in 100 macrophage cells was counted, and the IC 50 was determined (19,23).

Macrophage Cytotoxicity Assay
Since macrophage cells are the principal resident cells for Leishmania parasites, the J774A.1 macrophage was used in this study to assess the toxicity of ethyl acetate and methanol extracts and F1-F4 fractions of F. tabasensis at concentrations of 1.56 -200 µg/mL, alone or in combination with 200 µg/mL of ShCE.For this purpose, 2 × 10 5 cells/well were cultured in 96-well microplates with RPMI-1640 medium, containing 1% Pen-Strep and 12% FBS, and the cells were allowed to attach at a temperature of 37°C in a 5% CO 2 atmosphere for 12 hours.Next, 100 µL of each concentration was added to the wells and incubated for another 72 hours.Finally, cytotoxicity was determined Iran J Pharm Res.2023; 22(1):e136173.using the MTT assay, as described in the section above.Additionally, 50% cytotoxicity concentrations (CC 50 ) of the extracts and fractions were determined as a 50% reduction in the cell viability of treated cells relative to untreated cells.The ratio of CC 50 for macrophage cells to IC 50 for amastigotes was defined as the selectivity index (SI) (13, 19).

Flow Cytometry
The flow cytometry method was utilized to indicate apoptotic and necrotic cells using Annexin V Apoptosis Detection Kit (MabTag, Germany), according to the manufacturer's instructions.In brief, 1 × 10 6 logarithmic-phase promastigotes, 1 × 10 5 uninfected macrophages, and 200 µL of infected macrophage cells, exposed to the IC 50 concentrations of extracts and fractions, were collected.They were first washed with a cold, sterile PBS solution and then centrifuged for 15 minutes at 1000 g.Next, 5 µL of annexin V and propidium iodide (PI), as well as 400 µL of binding buffer, were added to the cell pellets.The promastigotes and macrophage cells were incubated at 25°C and 37°C, respectively, in a dark room for 15 minutes.The test results were read in a CyFlow Space Flow cytometer (Sysmex-Partec, USA), and the collected data were analyzed in FlowJoTM Version 10.5.3 (Vancouver, BC, Canada).All the experiments were performed in triplicate, and the percentages of apoptosis and necrosis were assessed for each tested sample.

Statistical Analysis
Data are presented as mean and standard deviation (SD) of experiments run in triplicate.The IC 50 values of promastigotes and amastigotes, as well as the CC 50 values of J774A.1 macrophage cell were calculated based on the mean viability percentage of promastigotes, amastigotes, and macrophage cells, respectively, against the untreated controls.Microsoft Excel Version 16 and SPSS Version 24 (SPSS Inc., Chicago, IL, USA) was used for evaluating the results, drawing figures, and analyzing the data.Moreover, the repeated measures ANOVA test was used to analyze the results of the parasite counting method.A one-way ANOVA test, followed by Tukey's post-hoc test, was also performed to compare the IC 50 and CC 50 values of compounds.Finally, the SI value was calculated by determining the ratio of CC 50 to IC 50 for amastigotes.

Parasite Counting Method
According to the microscopic analysis, the antileishmanial activity of F. tabasensis extracts and fractions against L. major promastigotes varied in a dose-and time-dependent manner.The number of promastigotes was markedly reduced by increasing the concentrations of extracts and fractions and also over time (from 24 to 72 hours) compared to the negative control (CTRL-) (P < 0.05).The F1 and methanol extracts were more effective against promastigotes compared to the ethyl acetate extract and other fractions (Table 1).Overall, the present results highlighted the remarkable effects of ShCE at a concentration of 200 µg/mL in combination with the IC 50 concentrations of F. tabasensis extracts and fractions against the promastigotes (P < 0.05) (Table 2).

MTT Assay for Promastigote Viability
Based on the results, the viability of promastigotes against various concentrations of extracts and fractions (1.56 -200 µg/mL) decreased in a dose-dependent manner and efficacy increased with an enhancement of drug concentrations.In other words, concentrations of 1.56 and 200 µg/mL resulted in the highest and lowest viability percent, respectively (P < 0.001).
The results of statistical analysis indicated significant differences between all the groups in comparison to the control group (P < 0.05).
The results of statistical analysis indicated significant differences between all the groups compared to the control group (P < 0.05).

Amastigote Assay
The results of the amastigote assay revealed that different concentrations of F. tabasensis extracts and fractions resulted in a remarkable reduction in both the number of infected macrophages and the number of intra-amastigotes in comparison to the negative control (Ctrl-) (P < 0.05).Overall, 42.5% of J774A.1 macrophage cell was infected in the Ctrl-group.Concerning the effects of exposure to the extracts and fractions, it was found that the ethyl acetate and methanol extracts, F1 fraction, and F3 fraction significantly decreased the number of infected macrophage cells, as well as the mean number of intracellular amastigotes at different concentrations compared to the other studied compounds and positive controls (P < 0.001) (Table 3).Interestingly, the IC 50 was lower than the IC 50 calculated for the promastigotes, except for the F2 fraction (Table 4).On the other hand, ShCE at 200 µg/mL, combined with various concentrations of extracts or fractions, significantly reduced the number of infected macrophages, as well as the mean number of intra-amastigotes (P < 0.001); therefore, its combined use with F. tabasensis extracts and fractions was more efficient than its independent use (data are not shown).
The results of statistical analysis revealed significant differences between all groups compared to the control group (P < 0.05).

Cytotoxicity of Macrophages
Similar to promastigotes and amastigotes, the cytotoxicity of macrophages was based on the concentrations of extracts and fractions (P < 0.05).Evaluation of all extracts and fractions, except F2, indicated the low toxic effects of macrophages (SI>10).As shown in Table 4, the highest and lowest IC 50 values were 295 ± 15.2 and 19.5 ± 2.6 µg/mL, attributed to the F4 and F2 fractions, respectively (Table 4).

Flow Cytometry
The exposure of stationary-phase L. major promastigotes, as well as infected and non-infected macrophages, to the extracts and fractions of F. tabasensis at their IC 50 concentrations after 72 hours induced early apoptosis (Q3, positive for Annexin V), late apoptosis (Q2, positive for Annexin V and PI), necrosis (Q1, positive for PI), and alive cells (Q4, negative for Annexin V and PI) compared to the untreated control cells.The percentage of apoptotic and necrotic cells varied depending on the extract or fraction.
As shown in Figure 2 -4, significant early apoptosis of promastigote cells was detected in exposure to the methanol extract and F1 fraction.
Additionally, the results of flow cytometry indicated the synergistic effects of extracts and fractions when combined with ShCE.Interestingly, the percentage of apoptosis (early and late apoptosis) was higher in the infected macrophage cells compared to the promastigotes and non-infected macrophages.There were significant differences in terms of early and late apoptosis and also necrosis of all extracts and fractions only or combined with ShCE compared to the negative control (CTRL) (P < 0.001).

Discussion
The results of the present study revealed that the ethyl acetate and methanol extracts and F1-F4 fractions of F. tabasensis had potential activities, alone or synergistically with ShCE, against both L. major promastigotes and intra-macrophage amastigotes in a dose-dependent manner.The IC 50 values for the promastigotes were 3.8 ± 1.13, 2.9 ± 0.55, 2.4 ± 0.29, 4.85 ± 1.2, 4.27 ± 1.82, and 33.5 ± 2.66 µg/mL in the ethyl acetate and methanol extracts and F1-F4 fractions, respectively.The corresponding values for GLU and AmB, as the reference drugs, were 420 ± 1.9 and 33.9 ± 5.1 µg/mL, respectively.

Since macrophage cells are the main host cells for
Leishmania parasites, one of the most important steps in the fight against Leishmania parasites is to prevent the 6 Iran J Pharm Res.2023; 22(1):e136173.tabasensis used alone or in combination with ShCE infectivity of macrophage cells and destroy them (29).The present findings showed the inhibitory effects of all extracts and fractions against intracellular amastigotes of L. major as the main causative agent of Old World CL.The IC 50 value for amastigotes was lower than that of promastigotes (except F2).Based on the results, the combination of ShCE at 200 µg/mL with Ferula extracts and fractions at IC 50 concentrations was more efficient than either of the drugs used alone (P < 0.001).However, the toxicity of non-infected macrophage cells was low, and the SI values were > 10 (except F2), representing a safety index for the application of these extracts and fractions to eliminate infected macrophages.
The strong antileishmanial activities of sesquiterpenes (50,51), monoterpenes, sulfur-containing compounds (51), and volatile terpenoids from the genus Ferula (48) have been described in the literature.In our previous research, we found that in vitro exposure of promastigotes to ShCE has significant effects, including a reduction in the growth rate and viability of promastigotes, besides synergistic effects with artemisinin on both promastigotes and amastigotes in vitro and in vivo (21).The present results, for the first time, revealed that both extracts and fractions of F. tabasensis, combined with ShCE, exerted enhanced leishmanicidal effects against L. major.
Flow cytometry is an alternative technique for determining the type of programmed cell death, including early and late apoptosis and necrosis, and also for examining the effects of extracts or fractions on viability or mortality (52).It has been indicated that Leishmania prevents the apoptosis of infected macrophage cells.On the other hand, apoptosis occurs in Leishmania amastigotes and promastigotes following exposure to drugs and herbal extracts (53).In the present study, the results of flow cytometry confirmed the promastigote and amastigote assay results, which suggested significant apoptosis at IC 50 concentrations of all extracts and fractions.The percentage of apoptosis (early and late) of promastigotes following exposure to ethyl acetate and methanol extracts and F1-4 fractions of F. tabasensis was measured to be 18.44%, 44.2%, 43.83%, 29.09%, 29.05%, and 11.3%, respectively; these values also increased when the extracts and fractions were combined with ShCE.
In this regard, Gharaei et al. reported the apoptosis-inducing effects of F. gummosa Boiss extracts in AGS, a human adenocarcinoma cell line.In this study, the ethanol extract from plant flowers induced high apoptosis (78%) in the promastigote cells (54).Moreover, in a study by Mousavi et al., the apoptotic effects of auraptene, as one of the key components of 7-prenyloxycoumarins from F. szowitsiana, were documented in the MCF-7 cell line (IC 50 = 59.7 µM).In this study, DNA fragmentation was introduced as one of the underlying mechanisms of component-induced apoptosis (55).

Conclusions
Owing to the potent antileishmanial activity of F. tabasensis extracts and fractions against L. major, especially the methanol extract and F1 fraction used alone or in combination with ShCE, they can be not only introduced as new drug alternatives in antileishmanial therapy, but also support future research for the development of highly effective, affordable, and reliable medicines.

Figure 2 .
Figure 2. The apoptotic and necrotic profiles of L. major promastigotes in exposure to IC50 concentrations of ethyl acetate and methanol extracts and F1-F4 fractions of F.

Figure 3 .Figure 4 .
Figure 3.The apoptotic and necrotic profiles of L. major promastigotes in exposure to IC50 concentrations of ethyl acetate and methanol extracts and F1-F4 fractions of F. tabasensis used alone or in combination with non-infected macrophages

Table 1 .
The Average Number of L. major Promastigotes (×10 4 ) in Exposure to Different Concentrations of F. tabasensis Extracts and Fractions After 24, 48, and 72 Hours (n = 3) a Data are expressed as mean ± SD.

Table 4 .
Comparison of IC50 Values of Ethyl Acetate and Methanol Extracts and F1-F4 Fractions of F. tabasensis for Promastigotes, IC50 Values for Amastigotes, and CC50 Values for the J774A.1 Macrophage Cells