Effect of Foeniculum Vulgare Aqueous and Alcoholic Seed Extract against Zoonotic Cutaneous Leishmaniasis

Background Cutaneous leishmaniasis is considered one of the major neglected tropical diseases. Drug resistance, limitary efficacy, and severe side effects remain a challenge for treatment. Foeniculum vulgare is known as a medicinal plant belonging to the Apiaceae, and anti-microbial properties of this plant have already been confirmed. Method The F.vulgare sterile aqueous and alcoholic extracts were prepared. In vitro has used RAW 264.7 cell line and L. major parasite (MRHO/IR/75/ER). Cytotoxicity assay on macrophages (CC50), cytotoxicity assay on promastigotes (IC50), and cytotoxicity assay on infected macrophages (EC50) were accomplished with both extracts by MTT and light microscopy methods. Four in vivo were allocated in four groups and five BALB/c mice each group. Stationary phase promastigotes were inoculated into the base of mice tails subcutaneously (SC). Measurement of the body weight, lesion size, parasite burden of the lesion, and spleen after 4 weeks for evaluation effects of the alcoholic extract on CL was done. Results The results of in vitro revealed that the optimal concentrations of both extracts reducing the promastigotes and amastigotes growth. Alcoholic extract no harmful side effects for the host macrophages, while were indicated has a potent action against L. major. In vivo results after 4 weeks did not show any variation in lesion size and body weight. Also, lesion size and spleen parasite burden decreased in comparison to no treatment group. Conclusion The alcoholic extract could be a new alternative treatment for cutaneous leishmaniasis. However this extract needs more investigation for novel herbal drugs against CL.


INTRODUCTION
Leishmaniasis is considered one of the six major neglected tropical diseases (NTDs) by the World Health Organization (WHO) (1). Currently, 13 million people in 98 countries have different types of leishmaniasis (2), and according to a 2017 report from the World Health Organization, about 600,000 to 1 million new cases each year have become a public health hazard worldwide (https://www.who.int/newsroom/fact-sheets/detail/leishmaniasis). The most common form of leishmaniasis is the cutaneous form caused by the protozoan parasites belonging to the genus Leishmania (3,4). Drug resistance, limitary efficacy and severe side effects of common drugs such as sodium stibogluconate and meglumine antimoniate remain a challenge for treatment of cutaneous leishmaniasis (5). Therefore, these agents provide the need for a new effective antileishmanial drug.
Natural products and their compounds have long been used in the production of new drugs because they are cost-effective and easily accessible (6). Sweet fennel (Foeniculum vulgare) is known as a medicinal and aromatic plant belonging to the Apiaceae (7,8). Antimicrobial properties of Foeniculum vulgare extract have been confirmed on bacteria and fungi (9,10) so that it may affect the Leishmania parasite.
This is the first study that has been done on the anti-parasitic characteristics of F. vulgare aqueous and alcoholic seed extract against cutaneous leishmaniasis (CL) due to L. major. In in vivo, the most effective fennel extract, its cytotoxicity effects on promastigotes, macrophages and infected macrophages have been assessed. Lesion size and parasite burden of the spleen were measured in the infected BALB/c mice.

MATERIALS AND METHODS
Preparation of plant extract: The F. vulgare seeds were obtained from the Barij Essence Pharmaceutical Company (Iran). Plant seeds were ground and macerated. Twenty-five grams of powder was solvated with 200 ml methanol or distilled water and was shaken for 72 hours at 25°C. After filtration through whatman filter paper (No. 4), re-extraction was carried out two times, and then the extracts of every sample were evaporated (25°C) and dehydrated in desiccators under vacuum to a constant weight. At the end, products were sterilized entirely by filtration with a 0.22 µm membrane filter. Finally, the alcoholic and aqueous extracts were applied in the project freshly. In-vitro cytotoxicity assay of the alcoholic and aqueous extracts on macrophages (CC50): In order to determine the cytotoxicity dosage of F. vulgare extract, the RAW 264.7 cells (1×10 5 cells) were seeded in the presence of the extract growing concentrations (10 to 4500 µg/ml) in 96-well microliter culture plates in 5% CO2 for 48 h at 37 ˚C. Cell viabilities were measured using colorimetric 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich, Deisenhofen, Germany) assay as it was described earlier (11). No treatment and glucantime® (5 mg/mL) were used as controls. Absorbance ratios were measured at 530 nm wavelength. These results were indicated as the mean percentage (%) reduction of macrophages in comparison with untreated control samples × 100. Finally, the concentration producing 50% (µg/ml) (CC50) was determined with respect to the method performed by Weislow et al. (12). CC50 amounts were DOI: http://dx.doi.org/10.4314/ejhs.v31i2.23 obtained using Prism 8 software (Graph-Pad Prism, San Diego, California, USA).

In-vitro cytotoxicity assay of the extracts on promastigotes (IC50):
The effects of rising concentrations of F. vulgare extract (10 to 4500 µg/ml) on the stationary phase of L. major promastigotes (1×10 6 parasites) were quantified for 48 hours at 26 ˚C. The susceptibility was determined by counting with Neubauer chamber (13) and the extract concentration was estimated, which resulted in 50% inhibition in promastigotes growth (µg/ml) (IC50).

In-vitro cytotoxicity assay of the extracts on infected macrophages by Leishmania (EC50):
RAW 264.7 macrophages (2×10 6 cells) were cultured onto a crystal chamber slide, along with in 24-well culture plate in DMEM medium supplemented with 10% FBS, which was incubated for 24 hours in 5% CO2 at 37˚C. L. major promastigotes were cultured at 25˚C in RPMI-1640 with 10% FBS to reach stationary growth phase. Then, to infect RAW 264.7 macrophages, promastigotes were added to each chamber slide and 24-wells at the ratio of 1:10 with parasite-to-host, followed by incubation for 24 hours. Free promastigotes were removed by washing two times using serum-free RPMI-1640 medium. Finally, infected macrophages were treated with increasing concentrations (10 to 4500 µg/ml) of F. vulgare extract for 48 hours at 37 ˚C in 5% CO2 as it was described earlier.
After 48 hours, RPMI 1640 medium was removed and 50 µL PBS was added. Then, it was scraped by scraper and transfered to slide. At the end of this stage, the slide was stained with Giemsa. The antileishmanial effect of the F. vulgare extract was assessed by microscopic method. Hundred macrophages were counted, and infected macrophage was reported as percent.
Selectivity Index (SI) Determination: Herein, the ratio of CC50 value of the cytotoxic activity to EC50 value of the antileishmanial activity was determined; then, the extract SI was calculated (14). Furthermore, SI of promastigotes was calculated (SI = CC50 Macrophages/IC50 promastigote) (15). SI value under 10 shows ideal antileishmanial activity for this compound. Briefly, the ideal herbal compounds have the low cytotoxic, and they have the high antileishmanial activity (higher reported values = greater extract activity) (14). Mice: All efforts were accomplished to decrease BALB/c mice suffering throughout the experiment period. All the mice were female of 5±1 week's old or 20g weight that were purchased from Iran Pasteur Institute. Mice were placed in plastic cages and with free access to enough food and drinking water. BALB/c mice were kept in a controlled animal care facility including 21-25˚C, humidity 57±2%, and 12 hours of light-dark cycles.

Parasite strain and mice infection: The
Leishmania parasite was sustained in a high virulent state throughout a continuous passage in susceptible BALB/c mice. The spleen tissues from infected BALB/c mice were squished and cultured at 26˚C in RPMI 1640 medium supplemented with 10% FBS containing 1% pen/strep. All 20 laboratory mice were inoculated subcutaneously (SC) into the tail base, with Leishmania promastigotes (2×10 6 ) at liquid stationary phase culture. After thirty days, nodules and lesions emerged on 20 inoculated mice.

Treatment of infected mice with F. vulgare alcoholic extract:
To test the F. vulgare extract effects on infected BALB/c mice, all mice were distributed into four separate groups: A, B, C, D, (five mice per each group). Group A was control group (received no treatment); group B received F. vulgare intralesionally (IL) according to in vitro (300µL; 4500 µg/mL); group C received glucantime® intralesionally (IL) (injections of 20 mg/kg); group D received PBS as placebo (three times per week for 4 weeks).

Measurement of the lesion size and mice body weight:
Size of the lesions and mice body weight was measured before and after 4 weeks of treatment using a metric caliper (Mitutoyo, Taiwan) and lab balance (Ohaus, Switzerland), respectively. Quantification of Leishmania parasite load in lesion region: To measure the parasite load of the lesions, sampling was performed from the margins of lesions by using a vaccinostyle, and then smears were prepared. Smears on glass slides were fixed with absolute methanol, stained with Giemsa. Slides were evaluated for the presence of amastigotes using Zeiss light microscope (Carl Zeiss, Germany). Quantification of Leishmania parasite load using limiting dilution assay (LDA): After eight weeks from infection, the Leishmania parasite load in the spleen was quantified by LDA. Furthermore, five mice from each group were scarified. Then, spleen was ground by 100 µm cell strainer and was transferred into the culture media;, amastigotes were transformed to the motile active promastigotes. Finally, motile promastigotes were counted by invert microscopy and parasite load was assessed throughout, limiting dilution assay test as it was described earlier (16).

Statistical analysis:
Statistical analyses of all cytotoxicity assays including CC50, EC50, and IC50 were performed by the use of Prism 8.0 software. Both one-way ANOVA and Student's t-test were used. Furthermore, statistical differences were supposed significant at pvalues less than 0.05. It is worth mentioning that all tests of our study were accomplished in duplicate. Figure 1, F. vulgare extract was only toxic for RAW 264.7 macrophages at high extract concentrations. The CC50 (µg/ml) values of F. vulgare alcoholic and aqueous extracts at 48 hours were 4500 µg/ml and 3900 µg/ml, respectively.

Figure 1: Cytotoxicity assay (CC50) of concentrations of F. vulgare alcoholic and aqueous extracts on mouse RAW 264.7 macrophage cell line after 48 hours using MTT method in vitro
Antileishmanial effects of F. vulgare extracts on Leishmania promastigotes (IC50): The F. vulgare alcoholic and aqueous extracts strongly inhibited the Leishmania parasite growth with the IC50 of 1200µg/ml and 2500 µg/ml, respectively at 48 hours ( Figure 2). Parasite Rescue and Transformation Assay (PRTA or transformed promastigotes) (EC50): As indicated in Figure 3, the F. vulgare alcoholic and aqueous extracts could inhibit the growth of Leishmania amastigotes, EC50 3900 µg/ml and EC50 4250 µg/ml, respectively ( Fig  3). Therefore, F. vulgare extract did not indicate any negative/toxic effects on mice RAW 264.7 macrophage cell line, but it could inhibit the intracellular amastigotes growth and kill the parasite (Figures 1 and 3).

F. vulgare extracts and selectivity index (SI):
F. vulgare alcoholic and aqueous extracts were active against L. major amastigotes (1.15 µg/ml and 0.9 µg/ml, respectively) and promastigotes (3.75 µg/ml and 1.56 µg/ml, respectively) of L. major with a favorable SI compared to RAW 264.7 cell line macrophages. SI results were shown that alcoholic extract was more effective than aqueous extract.

Lesion size in infected BALB/c mice treatment with F. vulgare alcoholic extract:
To measure the effect of F. vulgare alcoholic extract on the experimental CL development, BALB/c mice were infected with stationary phase L. major. Afterwards, the development of CL lesion was monitored 8 weeks. Although CL lesions size in group B had not significant variation before and after treatment with F. vulgare alcoholic extract, it was reduced significantly as compared with A and D groups (Table 1) (p<0.05). Even if body weight of BALB/c mice in treated group with F. vulgare alcoholic extract (B group) had not considerable differences with prior treatment, body weight of this group increased significantly in comparison with the control group(A group) (p<0.05) ( Table  2).

DISCUSSION
Traditionally, attempts have been made to use herbal plants to treat cutaneous leishmaniasis (17). As previously mentioned, resistance to chemical drugs and their side effects have been reported in the treatment of cutaneous leishmaniasis (5). Herbal useful remedies could be used to replace chemical treatments for cutaneous leishmaniasis treatment (18). F. vulgare, belonging to the Umbelliferae (Apiaceae) family, is a plant native to the Mediterranean area. Fennel are cultivated in different regions of Europe and Asia, and most of them are imported from India, China and Egypt (19)(20)(21). Various studies have reported on the effect of this plant extract on genital system, respiratory disorders and digestive system (22,23). F. vulgare is known as anti-microbial, anti-inflammatory, anti-diabetic, anti-tumor, with low toxicity and has immunomodulatory effect (9,22,24,25). This study showed the therapeutic effects of aqueous and alcoholic extracts of F. vulgare as herbal drug without extreme toxicity to host cells. Determination of anti-leishmanial activity of F. vulgare extracts were accomplished with IC50, CC50 and EC50. The results of this research revealed that the optimal concentrations of the alcoholic extract for reducing the promastigotes and amastigotes growth were 1200 and 3900 µg/ml, respectively. According to this study, F. vulgare alcoholic extract has a potent action against L. major promastigotes and amastigotes whereas it doesn't have any harmful side effect on the host macrophages. Two main components including bis (2-ethylhexyl) phthalate and trans-anethole, are predominantly responsible for most of antileishmanial effects (26). Kataoka et al. showed that the fennel extract could inhibit inflammatory event (27). The suppressive effects of methanol extract against acute and subacute sickness, type 4 allergic reactions through cyclooxygenase and lipoxygenase inhibition have already been confirmed (28).
There are active compounds with anti-microbial activity such as oleic acid and coumarin in aqueous and alcoholic extracts (29,30). The prenylated coumarins auraptene, galbanic acid and umbelliprenin isolated from another genus Apiaceae (Ferula szowitsiana) were revealed invitro antileishmanial effects on L. major promastigotes in comparison with the negative control (31,32,33). The SI was known as a major index of herbal extract activity that is used in order to describe a compound's in vitro DOI: http://dx.doi.org/10.4314/ejhs.v31i2.23 efficacy in the parasite proliferation inhibition (15). This index revealed that the F. vulgare alcoholic extract was more toxic to L. major parasite in comparison with the macrophages. Indeed, the alcoholic extract indicated a remarkable anti-leishmanial activity, and has the potency to decrease the survival rate of amastigotes at nontoxic concentrations for the host cell. After treatment of infected BALB/c mice with F. vulgare alcoholic extract, all of the treatment groups did not show any increase in lesions size in comparison with no treatment and PBS groups. The LDA and light microscopy method demonstrated a considerable difference between the parasite burden in the F. vulgare extract and a glucantime® treated groups, in comparison with PBS and no treatment. Also, a significant increase in body weight of F. vulgare alcoholic extract treated mice showed that this extract had no toxic side effects on mice growth. In this study, methanolic extract is more effective than aqueous extract to killing Leishmania parasite but toxic for mammalian cells. As shown in Hamdy Roby and et al study, using other solvent such as hexane and diethyl ether can be found having better result against Leishmania major (10). Many studies have been conducted on effective drugs in order to decrease infection by parasites. Some research has been done on the use of herbal drugs on L. major parasites. Various types of drug delivery and nanoparticle therapy were examined in order to determine the effectiveness of herbal materials and disease treatments by pharmaceutical companies. In Iran, different types of herbal extracts have been considered for treating the L. major infection. Previously, no evidences reported on the anti-leishmanial effects of F. vulgare alcoholic extract in a mice model. This herbal extract could be a new alternative treatment for cutaneous leishmaniasis. Therefore, F. vulgare alcoholic extract with more investigation could be a candidate for novel herbal drugs against neglected tropical diseases such as leishmaniasis, and its promising effects for future studies is considerable.