Therapeutic potential of hesperidin methyl chalcone in the experimental treatment of cutaneous leishmaniasis

Potencial terapêutico da hesperidina metil chalcona no tratamento experimental da leishmaniose cutânea

ABSTRACT

Drug repositioning seeks to discover new applications for a drug that has already been approved in the market, resulting in faster and lower cost solutions, generally used for diseases that receive little investment, such as cutaneous leishmaniasis. In this context, hesperidin, commercially approved as Daflon^®, is a flavonoid that belongs to the chalcones group, a class that has antileishmanial potential. The present study evaluated the in vitro and in vivo antileishmanial activity of commercial hesperidin. Hesperidin (9 to 0.56 mg mL^-1) was tested against promastigote and amastigote forms of four dermotropic species of Leishmania, namely L. (L.) amazonensis, L. (V.) guyanensis, L. (V.) braziliensis and L. (V.) naiffi. For the in vivo tests, hamsters were infected in the snout and the lesions were treated with intralesional hesperidin. The treatment effectiveness was assessed by measuring the total volume of the lesion on the snout and determining the parasitic load. The in vitro results showed moderate toxicity in murine macrophages, with higher efficacy in L. (L.) amazonensis when compared to the other species tested. The in vivo results showed that hesperidin was able to gradually reduce the size of lesions by L. (L.) amazonensis, although it did not induce clinical and parasitological cure. Thus, hesperidin showed potential in in vitro tests against L. (L.) amazonensis and further studies with new formulations and experimental treatment schemes should be carried out.

KEYWORDS: flavonoids; Leishmania; drug replacement; preclinical study

RESUMO

O reposicionamento de medicamentos busca descobrir novas aplicações para um medicamento já aprovado no mercado, resultando em soluções mais rápidas e de menor custo, geralmente utilizadas para doenças que recebem pouco investimento, como a leishmaniose tegumentar. Nesse contexto, a hesperidina, aprovada comercialmente como Daflon^®, tem despertado interesse científico por ser um flavonoide pertencente ao grupo das chalconas, classe que possui potencial antileishmania. O presente estudo avaliou a atividade antileishmania in vitro e in vivo da hesperidina comercial. A hesperidina (9 a 0,56 mg mL^-1) foi testada contra as formas promastigota e amastigota de quatro espécies dermotrópicas de Leishmania, sendo elas L. (L.) amazonensis, L. (V.) guyanensis, L. (V.) braziliensis e L. (V.) naiffi. Para os testes, os hamsters foram infectados no focinho e as lesões tratadas com hesperidina intralesional, cuja eficácia foi avaliada através da aferição do volume total da lesão no focinho e a determinação da carga parasitária. Os resultados in vitro demonstraram toxicidade moderada em macrófagos murinos e melhor eficácia em Leishmania (L.) amazonensis quando comparados às demais espécies testadas. Os resultados in vivo mostraram que a hesperidina foi capaz de reduzir gradativamente o tamanho das lesões causadas por L. (L.) amazonensis, embora não tenha induzido a cura clínica e parasitológica. Assim, a hesperidina mostrou-se promissora em estudos in vitro contra Leishmania (L.) amazonensis e novos estudos com novas formulações e esquemas de tratamento experimental devem ser realizados.

PALAVRAS-CHAVE: flavonóides; Leishmania; substituição de drogas; estudo pré-clínico

INTRODUCTION

Cutaneous leishmaniasis (CL) is a tropical, infectious-parasitic and non-contagious disease caused by the protozoa of the genus Leishmania. It presents itself as a polymorphic manifestation of the skin and mucosa, and is associated with high morbidity (Blanco and Nascimento-Júnior 2017; Glans et al. 2018). In Brazil, there are seven pathological species that cause CL, six of the subgenus Viannia and one of the subgenus Leishmania, namely L. (V.) braziliensis Vianna, 1911, L. (V.) guyanensis Floch, 1954, L. (V.) lainsoni Silveira, Shaw, Braga & Ishikawa, 1987, L. (V.) naiffi Lainson & Shaw, 1989, L. (V.) shawi Lainson, Braga & de Souza, 1989, L. (V.) lindenbergi Silveira, Ishikawa & de Souza, 2002 and L. (L.) amazonensis Lainson & Shaw, 1972 (Teles et al. 2016).

As recommended by the Brazilian Ministry of Health, the treatment of the different clinical forms of CL is carried out via the use of five drugs, namely meglumine antimoniate, pentamidine isethionate, amphotericin B or liposomal amphotericin B and the recently included miltefosine (Brasil 2017; SVS 2020), but there are therapeutic limitations due to low efficacy, high toxicity, long and painful treatment, and high financial costs, addition, parasites may be resistant to treatment (Brasil 2017). Given these limitations, the World Health Organization recommends the search for new medicines and treatments for CL (WHO 2021).

One path in the search for new treatments is drug repositioning, which aims to investigate whether a drug that has already been approved and is used clinically has some activity against another disease that is still untreated (Xue et al. 2018; Jourdan et al. 2020). This strategy has generally been used for diseases that receive little investment, such as CL (Bustamante et al. 2019). In this context, some flavonoids have aroused interest in the scientific community. Among them is hesperidin, a substance that belongs to the group of chalcones, which has been approved in clinical therapy and is commercially known as Daflon^®, and is used in synergism with diosmin for the treatment of varicose veins and other symptoms related to chronic venous insufficiency (Servier 2019). Hesperidin has been investigated for its anticancer (Pandey et al. 2021), antioxidant (Hager-Theodorides et al. 2021), antiviral (Attia et al. 2021), immunomodulatory (Berkoz et al. 2021) activity, and as a treatment for visceral leishmaniasis (Tabrez et al. 2021a).

There are no studies in the literature that investigate the repositioning of hesperidin for the treatment of CL. Nonetheless, Tabrez et al. (2021a) showed results of hesperidin activity in promastigote (IC₅₀ 1.019 mM) and amastigote (IC₅₀ 0.285 mM) forms of Leishmania donovani (Laveran & Mesnil, 1903) and studied its possible mechanism of action. Hesperidin (at a concentration of 2.0 mM) induced reactive oxygen species in 96.7% of the parasites in a dose-dependent manner and consequent induction of apoptosis-like cell death, together with the possible inhibition of a key ergosterol-biosynthetic enzyme (Tabrez et al. 2021a).

In this study, we evaluated the antileishmanial activity of hesperidin in vitro and in vivo against different species of Leishmania that cause CL in Brazil.

MATERIAL AND METHODS

Substances

The target molecule under study was hesperidin methyl chalcone, which was purchased commercially (Sigma Aldrich^TM, São Paulo, Brazil), and meglumine antimoniate (Glucantime^®; Sanofi-Aventis, São Paulo, Brazil), which is the standard drug used as the positive control.

Parasitic mass

We used strains of Leishmania (Leishmania) amazonensis (IFLA/BR/1967/PH8), Leishmania (Viannia) guyanensis (MHOM/BR/1975/M4147), Leishmania (Viannia) braziliensis (MHOM/BR/1975/2904) and Leishmania (Viannia) naiffi (MDAS/BR/1979/M5533) maintained and cryopreserved in the Laboratory of Leishmaniasis and Chagas Disease at Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus, Brazil. The strains were initially cultivated in Novy-MacNeal-Nicolle (NNN) medium and the expanded culture in Roswell Park Memorial Institute medium (RPMI 1640 - Sigma Chemical Co. St. Louis, USA), which was supplemented with 10% inactivated fetal bovine serum (iFBS) (LGC Biotecnologia, São Paulo, Brazil) and 50 µg.mL^-1 of gentamicin (Novafarma, Brazil), and incubated at 25 ºC.

Ethical approval

This study was approved by the ethics committee on the use of animals at INPA for the performance of the in vitro and in vivo tests (authorization # 029/2020 CEUA/INPA).

Cytotoxicity assay

Murine peritoneal macrophages were grown in 96-well plates at a concentration of 10⁵ cells mL^-1 in RPMI medium without the Ph/phenol red indicator (Sigma Chemical Co. St. Louis, USA), supplemented with 10% iFBS and kept in an incubator at 5% CO₂ (Form Series II Water Jacket CO₂ Incubator, Thermo Scientific, USA) and 37 °C for 24 hours. Cells were treated with different concentrations of hesperidin (9.00, 4.50, 2.25, 1.12 and 0.56 mg mL^-1) and Glucantime^® (8.00, 4.00, 2.00, 1.00 and 0.50 mg Sb⁵⁺ mL) distributed on each plate in triplicate and five contractions. Wells with untreated cells and wells without cells were used as controls. The hesperidin concentrations followed Gomes et al. (2017).

Cell viability was evaluated by the colorimetric method using Alamar Blue^® (Sigma Aldrich™, USA) at 24, 48 and 72 hours (one plate per period of time) with the addition of 10 µL of sodium resazurin salt stock solution (4 mg.mL^-1 in phosphate-buffered saline solution) in each well. The plates were again incubated for another 12 h at 37 °C, after which they were read using a spectrophotometer (Elx800™, BIO-TEK^®, Winooski, Vermont, USA) at 590 nm (Chagas et al. 2021).

To evaluate cytotoxic activity, murine peritoneal macrophages were quantified in a Neubauer chamber, adjusted to 10⁵ cells mL^-1 and then incubated in a 24-well plate (three plates) against hesperidin at pre-established concentrations, kept under study at 37ºC with 5% CO2, in order to evaluate the morphology of the macrophages against the substances under test over a period of 24 to 72 hours.

Biological assay with promastigote forms

Promastigotes (2 x10⁶ promastigotes mL^-1) of the species under study were added to 96-well plates (one plate per species) and exposed to hesperidin and Glucantime^® distributed on each plate in triplicate and five concentrations as described for the cytotoxicity assay. Wells with untreated parasites were used as the negative control. Biological activity was determined by quantifying viable promastigotes at 24, 48 and 72 hours in a Neubauer chamber using an optical microscope (Nikon Eclipse E200, Japan) at 400x magnification. Data were expressed as the mean inhibitory concentration (IC₅₀) (Chagas et al. 2021).

Biological assay with amastigote forms

Murine peritoneal macrophages at a concentration of 10⁴ cells mL^-1 were cultured on glass coverslips inserted into 24-well plates (three plates for each species tested) with RPMI culture medium that was supplemented with 10% iFBS and infected with Leishmania spp. promastigotes at a concentration of 10⁵ promastigotes mL^-1, corresponding to the proportion of five promastigotes per macrophage for each species tested and then placed in an oven at 37 ºC with 5% CO₂ for two hours. Infected macrophages were treated with different concentrations of hesperidin (9.00, 4.50, 2.25 and 1.12 mg mL^-1) and Glucantime^® (8.00, 4.00, 2.00 and 1 .00 mg Sb⁵⁺ mL^-1) distributed on each plate in triplicate and four contractions for 24, 48 and 72 hours at 37 °C in 5% CO₂. Infected and untreated cells were used as negative control. Subsequently, the coverslips were fixed and stained every 24 hours using the Rapid Panoptic method (Laborclin^®, Paraná, Brazil) and analyzed using light microscopy. The percentage of infected cells was determined by randomly quantifying 100 infected and uninfected cells on each coverslip. Data were also expressed by the mean inhibitory concentration (IC₅₀) (Chagas et al. 2021).

In vivo biological assay

A total of 108 golden hamsters (Mesocricetus auratus Waterhouse, 1839), weighing approximately 180 g and aged 90 days, were used in this assay. This animal model was used due to its high susceptibility to dermotropic pathologies and the clinical evolution of the disease to present signs similar to those observed in humans. Furthermore, some of the species analyzed in the study have the capacity to evolve into oronasal infections (mucosal leishmaniasis), a manifestation that is difficult to treat in humans, which is why we chose to evaluate the progression of the disease/treatment in the snout region.

The animals were divided into three groups (A, B and C) of 36 animals. The animals were submitted to infection by inoculation in the snout of 0.1 mL^-1 of promastigotes (10⁶ promastigotes mL^-1) of L. (V.) guyanensis (Group A), L. (L.) amazonensis (Group B), L. (V.) braziliensis (Group C). Leishmania (V.) naiffi does not develop macroscopically visible skin lesions, therefore this species was not used in this assay. Each group was assigned to the following treatments/control (six animals per treatment/control): I) non-infected (GNI); II) infected and untreated (GINF); III) infected and treated intralesionally with saline solution (placebo) (GTP); IV) infected and treated intralesionally with 1.15 mg Sb⁵⁺ kg^-1 day^-1 of Glucantime^® (GTG); V) infected and treated intralesionally with 1.15 mg mL^-1 of hesperidin (GTH1); VI) infected and treated intralesionally with 4 mg mL^-1 of hesperidin (GTH2).

After the lesion appeared, the respective treatments were applied every 15 days, totaling 45 days of treatment (4 applications), according to the Brazilian Ministry of Health guidelines for intralesional treatment. After 45 days of treatment, the animals were observed (without receiving treatment) for another 15 days, thus totaling a follow-up of 60 days.

During the treatment, the total volume of the lesion was monitored weekly by measuring the snout derived average length, width and height using a digital caliper (Zaas precision^®, Brazil - 0.02 mm precision), photo-documentation of the snout for macroscopic morphological analysis of the lesion, and weighing of the animals for observation of weight gain or loss (Comandolli-Wyrepkowki et al. 2017). Monitoring was carried out by measuring the snout to monitor, in addition to the lesion, the formation of edema called “tapir nose”, characteristic of this pathology.

At the end of the experimental period, all animals were anesthetized (ketamine and xylazine) and euthanized intramuscularly. After euthanasia, biological material was collected (fragments of the lesion, liver, spleen, kidneys and blood), and the organs were weighed on precision scales (Shimadzu^®, Brazil - 0.1 mg precision) (Comandolli-Wyrepkowki et al. 2017).

Parasitological and hematological evaluation

The tissue fragments collected from the lesion area of ​​the animals were used to make prints on glass slides, which were later stained using the Rapid Panoptic method. Quantification of infected cells was determined by counting 25 fields at random under an optical microscope (Nikon Eclipse E200, Japan) at 1,000x magnification.

Lesion fragments and the liver were cultured in NNN medium for 7 days at 25 °C and then the presence or absence of promastigotes was evaluated. Cultures were considered positive when at least one form of the parasite was isolated in the culture medium (Comandolli-Wyrepkowki et al. 2017).

Blood smears were made on slides and then stained using the Rapid Panoptic method. The analysis was performed by counting 100 cells per slide with differentiation and quantification of leukocyte cells under an optical microscope with a 400x magnification.

Statistical analysis

IC50 was obtained through linear regression using the number of living cells. A one-way ANOVA followed by Tukey’s test was used to assess the significance of the differences between the groups of the in vivo biological assay, at the 5% significance level. All statistical analyses was performed using the GraphPad Prism program version 6.0 for Windows (GraphPad Software, San Diego, CA)

RESULTS

Cytotoxicity assay

Hesperidin showed moderate toxicity to murine peritoneal macrophages, with an average cell viability of 75% at the highest concentrations (9.00, 4.5, and 2.25 mg mL^-1) and 90% at the lower concentrations (1.12 and 0.56 mg mL^-1) (Figure 1). Cell viability and morphology of cells treated with hesperidin varied little in relation to the control group with untreated cells, with the presence of reduced-sized cells and differences in cell delimitations being observed at the higher concentrations tested.

Figure 1
Cytotoxic effect of hesperidin (9.00-0.56 mg mL^-1, dotted bars) and Glucantime^® (8 mg Sb^5+.mL^-1, black bars) on murine peritoneal macrophages at 24, 48 and 72 hours, evaluated by cell viability. Different letters within periods indicate significant differences according to a Tukey test (p < 0.05).

Biological assay with promastigote forms

Hesperidin showed a lower IC₅₀ in L. (L.) amazonensis promastigotes at 24 hours (IC₅₀ < 0.56 mg), followed by L. (V.) naiffi at 72 hours (IC₅₀ < 0.56 mg), L (V.) guyanensis at 72 hours (IC₅₀ 2.93 mg) and L. (V.) braziliensis at 48 hours (IC₅₀ 3.7 mg) (Table 1).

Table 1
Mean inhibitory concentration (IC₅₀) of hesperidin and Glucantime^® in promastigote forms of Leishmania spp. Values are the mean ± standard deviation of three replicates.

Species	Period (hours)	Hesperidin	Glucantime®
L. (L.) amazonensis	24	< 0.5 ± 2.0	10.1 ± 4.9
	48	1.5 ± 3.2	0.56 ± 3.4
	72	2.9 ± 2.2	3.3 ± 4.9
L. (V.) guyanensis	24	9.2 ± 11.7	11.0 ± 9.7
	48	9.1 ± 8.5	10.1 ± 12.0
	72	2.9 ± 3.1	2.92 ± 32.8
L. (V.) braziliensis	24	4.2 ± 8.0	11.6 ± 5.8
	48	3.7 ± 12.2	6.3 ± 9.5
	72	4.1 ± 12.0	5.2 ± 4.9
L. (V.) naiffi	24	4.1 ± 7.6	5.7 ± 17.0
	48	1.3 ± 8.9	3.2 ± 14.3
	72	< 0.5 ± 17.0	0.3 ± 29.3

With L. (L.) amazonensis, hesperidin induced a reduction in parasite viability (> 50%), with a significant difference at all concentrations in relation to the controls at 24 hours, and for the concentrations 9.00, 4.50 and 2.25 mg mL^-1 at 48 and 72 hours (Figure 2a). With L. (V.) guyanensis, there was a significant reduction in parasite viability at 72 hours compared to the negative control (Figure 2b). With L. (V.) braziliensis, there was a significant reduction in parasite viability at all times when compared to control groups (Figure 2c). With L. (V.) naiffi, the reduction in parasite viability occurred at 48 and 72 hours in relation to the negative control (Figure 2d).

Figure 2
Hesperidin activity (9.00-0.56 mg mL^-1, striped bars) against the promastigote forms of L. (L.) amazonensis (A), L. (V.) guyanensis (B), L. (V.) braziliensis (C), L. (V.) naiffi (D) at 24, 48 and 72 hours, compared to the negative control (black bar) and Glucantime^® (8 mg Sb^5+.mL^-1, positive control, white bar). Columns represent the mean and bars the standard deviation. Different letters within periods indicate significant difference according to a Tukey test (p < 0.05).

Biological assay with amastigote forms

Hesperidin showed a lower IC₅₀ in L. (L.) amazonensis amastigotes at 72 h (IC₅₀ 3.06 mg), L. (V.) guyanensis at 24 hours (IC₅₀ 6.29 mg), L. (V.) naiffi at 72 hours (IC₅₀ 7.17 mg) and L. (V.) braziliensis at 48 hours (IC₅₀ 7.66 mg) (Table 2).

Table 2
Mean inhibitory concentration (IC₅₀) of hesperidin and Glucantime^® in amastigote forms of Leishmania spp. Values are the mean ± standard deviation of three replicates.

Species	Period (hours)	Hesperidin	Glucantime®
L (L.) amazonensis	24	6.2 ± 1.9	10.7 ± 2.8
	48	6.7 ± 1.5	9.5 ± 1.2
	72	3.0 ± 1.0	9.9 ± 1.5
L. (V.) guyanensis	24	6.2 ± 2.8	9.5 ± 1.7
	48	7.4 ± 1.7	3.2 ± 5.1
	72	7.6 ± 2.1	4.2 ± 4.9
L. (V.) braziliensis	24	8.6 ± 2.8	> 8 ± 3.5
	48	7.6 ± 2.6	> 8 ± 2.1
	72	10.4 ± 1.7	> 8 ± 1.2
L. (V.) naiffi	24	12.3 ± 1.7	7.4 ± 3.8
	48	9.4 ± 1.7	9.2 ± 4.2
	72	7.1 ± 1.4	8.9 ± 2.8

As observed in the promastigote forms, hesperidin was more effective against L. (L.) amazonensis, inducing a reduction of > 50% in the infection rate at 9.00, 4.50 mg mL^-1 and > 40% at 2, 25 and 1.12 mg mL^-1 throughout the entire incubation period, but did not difer significantly from Glucantime^® (Figure 3a). Similar results were observed against L. (V.) guyanensis at 24 and 48 hours, L. (V.) braziliensis at all times and L. (V.) naiffi at 48 and 72 hours, not differing significantly from Glucantime^® (Figure 3b-d).

Figure 3
Effect of hesperidin (9.00-0.56 mg.mL^-1, striped bars) on intracellular amastigotes of L. (L.) amazonensis (A), L. (V.) guyanensis (B), L. (V.) braziliensis (C), L. (V.) naiffi (D) at 24, 48 and 72 hours after treatment with different concentrations of hesperidin, compared to the negative control (black bars) (infected and untreated macrophages), and Glucantime^® (8 mg.Sb^5+.mL^-1, white bars). Columns represent the mean and bars the standard deviation. Different letters within periods indicate significant differences according to a Tukey test (p < 0.05).

In vivo biological assay

The hamsters infected with L. (L.) amazonensis (GTH1) had a significantly lower weight (mean ± SD = 135.3 ± 5.2 g) than the other groups (172.1 ± 4.6 g). There were no significant differences in weight among groups in L. (V.) guyanensis and L. (V.) braziliensis assays. In the animals infected with L. (L.) amazonensis, there was a continuous reduction in the volume of the snout in GTH1 (56%) and GTH2 (49%), which did not differ significantly from GTG (57%) and GINF.

In animals infected with L. (V.) guyanensis and L. (V.) braziliensis, a reduction in the lesion volume was observed, on average, until the 45^th day of treatment and, after that, an increase was observed again in GTG, GTH1 and GTH2. At the end of the experiment, there was a significant difference for the GNI group (p < 0.001); however, there was no significant difference for the GINF group. At the end of treatment animals of the GTH1 group that were infected with L. (V.) guyanensis reduced the lesion volume by 41% and the GTH2 group infected with L. (V.) braziliensis reduced the lesion volume by 29% (Figure 4).

Figure 4
Snout volume of golden hamsters infected with L. (L.) amazonensis (A), L. (V.) guyanensis (B) and L. (V.) braziliensis (C). Black circles = uninfected and untreated - GNI (positive control); squares = infected and untreated - GINF (negative control); triangles = infected and treated with placebo - GTP (negative control); diamond = infected and treated with 1.15 mg Sb⁵⁺ kg^-1 day^-1 Glucantime^® - GTG (positive control); X = infected and treated with 1.15 mg mL^-1 hesperidin - GTH1; white circle = infected and treated with 4 mg mL^-1 hesperidin - GTH2.

Animals infected with L. (L.) amazonensis presented edema with an apparent lesion. In GINF and GTP, these signs were intensified from the 30^th day onwards, which resulted in a larger snout volume and ulcerated lesions at the end of the treatment when compared to the other experimental groups. After the 15^th day of treatment, GTH1 and GTH2 progressed to exacerbated ulcerated lesions, followed by the formation of eschars and the consequent reduction of the edema, without clinical cure. GTG showed healing from the 30^th day onwards and consequent clinical cure of the animals.

In the groups infected by L. (V.) guyanensis and L. (V.) braziliensis, the animals presented nodule formation with a small lesion and scabs, expressed in GINF, GTP and GTH1 throughout the experiment period. Only in GTG was there clinical cure of all animals. In GTH2 infected with L. (V.) braziliensis, half of the group showed clinical cure (Figure 5).

Figure 5
Aspect of lesions on the snout of golden hamsters, Mesocricetus auratus infected by L. (L.) amazonensis (A), L. (V.) guyanensis (B) and L. (V.) braziliensis (C) at the end of the 60-day experimental period. GNI - uninfected and untreated animal (positive control); GINF - infected, untreated animal (negative control); GTP - infected animal treated with placebo (negative control); GTG - infected animal treated with Glucantime^® (1.15 mg/Sb^5+/kg/day) (positive control); GTH1 - infected animal treated with 1.15 mg.mL^-1 of hesperidin; GTH2 - infected animal treated with 4 mg.mL^-1 of hesperidin. The columns represent the different species and the lines represent the treatment groups.s

Parasitological and hematological evaluation

After treatment parasite viability revealed the presence of viable flagellate parasites in the cultures of the lesion and liver fragments in all groups, and there was no significant difference in the infectivity rate between the treatment groups in all the species tested. Only GTG in L. (L.) amazonensis showed a significantly lower macrophage infectivity compared to GTH2 (Figure 6). Organ weight (liver, spleen and kidney) and leukocyte parameters did not differ significantly among the experimental groups in all species tested.

Figure 6
Macrophage infectivity rate quantified by printing on slides of lesion fragments from golden hamsters infected with L. (L.) amazonensis (A); L. (V.) guyanensis (B) and L. (V.) braziliensis (C). Infected and untreated animals (GINF); infected animals treated with 1.15 mg kg_-1 day_-1 of Glucantime^® (GTG); infected animals treated with placebo (GTP); infected animals treated with 1.15 mg mL^-1 of hesperidin (GTH1); infected animals treated with 4 mg mL^-1 of hesperidin (GTH2). Each column represents the mean and each bar the standard deviation of three replicates. Different letters within periods indicate significant differences according to a Tukey test (p < 0.05).

DISCUSSION

This study is an unprecedented report on the antileishmanial activity of hesperidin in CL. Due to the lack of information in the literature regarding its antileishmanial activity, the results obtained in this study will be presented from the perspective of the antileishmanial activity of hesperidin and other substances of the flavonoid class that have already been documented against a species that causes visceral leishmaniasis (VL).

In our study, hesperidin showed moderate cytotoxicity and cell viability (61.3% to 80%), which is similar to what was observed with flavonoids such as quercetin (50% to 100%) (Caetano et al. 2019) and cynaroside (20% to 50 %) (Tabrez et al. 2021b), yet FM09h (an amine-linked flavonoid) did not show cytotoxicity to macrophages (Chan et al. 2021).

In our antileishmanial activity tests, a dose-dependent response was observed in which the highest concentrations of hesperidin (9 and 4.5 mg mL^-1) were more effective in inhibiting promastigotes and amastigotes, similarly to the results by Gervazone et al. (2018) and Tabrez et al. (2021b). However, as different pathogen species respond differently to a drug, only the IC₅₀ and infection rate will assess whether the drug under study is showing antileishmanial biological activity or inducing cellular toxicity (Silva 2008).

Against L. (L.) amazonensis, hesperidin was able to inhibit promastigote forms (> 75%, IC₅₀ < 0.56 mg) and amastigote forms (> 50%, IC₅₀ 3.06 mg) after 72 hours, significantly higher compared to Glucantime^®. These results corroborate Chan et al. (2021) for the flavonoid FM09h against amastigotes (90%) and Gervazone et al. (2018) for the flavonoid 2HF against promastigotes (79%, IC₅₀ 20.9 μM) and amastigotes (90%, IC₅₀ 3.09 mM). Both latter studies used macrophages infected with L. (L.) amazonensis and observed the reduction of intracellular amastigotes without destruction of the macrophage.Against L. (V.) braziliensis, hesperidin was significantly more effective than Glucantime^® only against promastigotes (< 50%, IC₅₀ 3.7 mg), while Chan et al. (2021) and Caetano et al. (2019) describe an efficacy for both evolutionary forms against this species, with a significant difference in relation to the positive control.

Against L. (V.) guyanensis and L. (V.) naiffi, hesperidin was able to reduce the promastigote forms and the rate of infection by amastigotes, but did not differ significantly from Glucantime^®. As far as we know, there are no studies in the literature that evaluate the antileishmanial activity of a flavonoid for these species, so that this is the first report on antileishmanial activity of a flavonoid against these species.

As expected, the tested Leishmania species responded differently to in vitro treatment with hesperidin, however it is possible to observe similar response patterns by subgenus. The subgenus Leishmania showed higher susceptibility to hesperidin, with significantly higher eficacy than Glucantime^® in both evolutionary forms. Against Viannia, hesperidin also induced a reduction in both evolutionary forms, but did not differ significantly from Glucantime^®, with the exception of L. (V.) brasiliensis in promastigote form. The only other report of antileishmanial activity of hesperidin showed efficacy against promastigotes and amastigotes of L. (L.) donovani that causes VL (Tabrez et al. 2021a). As VL causes a disease with different clinical manifestations from those of CL, it is also expected that it responds differently to a drug. However, these data support the pattern of higher susceptibility of the subgenus Leishmania to hesperidin.

The results of the cytotoxicity essay in macrophages suggest that there may have been greater toxicity in terms of the cells being infected. Cells infected by different species of Leishmania are more susceptible to the cytotoxic effects of compounds than non-infected cells (Silva 2008), which may be owed to cell membrane alterations caused by the parasite (Quintana et al. 2010). It is thus important that further studies on antileishmanial activity of hesperidin investigate the mechanism of action of this substance in infected and non-infected cells to determine the cytotoxicity of the substance.

Currently, the drugs used in the treatment of CL are associated with numerous adverse side effects, including high toxicity, drug resistance and requires parenteral administration. It is considered a difficult treatment to administer (DNDi 2018) and intralesional treatment is preferable since it allows the direct delivery of drugs to the skin lesions, avoiding or minimizing the adverse effects of systemic therapy, which can lead to a reduction in toxicity (Franco et al. 2016; Nassif et al. 2017). Our results indicated that the animals treated with hesperidin did not achieve clinical cure (lesion healing) nor parasitological cure (absence of parasites) against L. (L.) amazonensis and L. (V.) guyanensis. Against L. (V.) braziliensis, there was clinical cure in half of the animals treated with the higher dosis of hesperidin, although there was no parasitological cure, while in animals treated with Glucantime^® clinical cure was achieved in all cases, but also no parasitological cure. According to the Brazilian Ministry of Health, clinical cure of CL occurs when re-epithelialization of the ulcerated lesions and the total regression of the infiltration and erythema are observed (Brasil 2017), thus hesperidin did not meet the official standard of clinical cure for CL against any of the species tested.

Few studies have evaluated the in vivo effect of flavonoids in the search for new treatment alternatives for CL. A promising antileishmanicidal effect against L. (L.) amazonensis in mice was observed for 2HF (50 mg kg^-1 day^-1 administered orally, with significantly higher efficacy compared to Glucantime^®) (Gervazone et al. 2018) and FM09h (10 mg kg^-1 day^-1 administered intralesionally) (Chan et al. 2021). No toxic reactions, nor significant changes in the weight and leukogram of the animals, or in the size, weight and appearance of their organs were observed in the latter studies, suggesting that these substances did not cause apparent toxicity to mice.

It is characteristic of the subgenus Leishmania to present a more exacerbated evolution of CL with extensive lesions, causing large ulcerations in some cases, while in the subgenus Viannia the presence of nodules with small and controlled lesions is more common (Brasil, 2017). This differential response to infection is reflected in our results, as the hamsters infected with L. (V.) brasiliensis were more responsive to treatment, resulting in clinical cure with Glucantime^® and partial clinical cure with hespiridin.

There is no validated animal model for in vivo tests with Leishmania, and consequently a diversity of animal models are used, such as golden hamsters (Chagas et al. 2021), C57BL/6 mice (Sampaio et al. 2003), BALB mice/C and CBA mice (Reis et al. 2006). Hamsters are among the most promising experimental models for the study of leishmaniasis, due to their high susceptibility to dermotropic pathologies and for presenting a clinical evolution with signs similar to those observed in humans (Dea-Ayuela et al. 2007).

In the in vivo studies, the subgenera of Leishmania showed differencial clinical evolution patterns in response to treatment, although different from what was observed in the in vitro tests. The large and ulcerated lesions with progressive evolution and little response to treatment in the subgenus Leishmania may have occurred due to the difficulty in controlling the size of the lesions, while the small and controlled lesions in the subgenus Viannia were comparatively more responsive to treatment, resulting in clinical cure with Glucantime^® against L. (V.) brasiliensis.

CONCLUSIONS

Our study revealed that hesperidin shows promising activity against promastigote and amastigote forms of L. (L.) amazonensis. However, in the in vivo tests with golden hamsters, the best result with hesperidin was the 50% induced clinical cure of animals infected with L. (V.) brasiliensis when treated intralesionally with 4 mg mL^-1. Further studies should evaluate the effectiveness of hesperidin using new experimental treatment schemes, with emphasis on concentrations of the active ingredient, treatment time, Leishmania species involved and routes of administration, while also seeking to elucidate the therapeutic mechanisms involved in the response against infection of the species that cause CL.

ACKNOWLEDGMENTS

The authors thank the Fundação de Amparo à Pesquisa do Amazonas - FAPEAM, MSc. Maricleide de Farias Naiff and laboratory technician Lorival Maciel Castro.

REFERENCES

• Attia, G.; Moemen, I.S.; Youns, M.; Ibrahim, A.M.; Abdou, R.; El Rey, M.A. 2021. Antiviral zinc oxide nanoparticles mediated by hesperidin and in silico comparison study between antiviral phenolics as anti-SARS-CoV-2. Colloids and Surfaces B: Biointerfaces, 203: 111724.
• Berkoz, M.; Yalin, S.; Ozkan-Yilmaz, S.; Ozkan-Yilmaz, A.; Krozniak, M.; Francik, R.; et al 2021. Protective effect of myricetin, apigenin, and hesperidin pretreatments on cyclophosphamide-induced immunosuppression. Immunopharmacology and Immunotoxicology, 43: 353-369.
• Blanco, V.R.; Nascimento-Júnior, N.M. 2017. Leishmaniose: Aspectos gerais relacionados com a doença, o ciclo do parasita, fármacos disponíveis, novos protótipos e vacinas. Revista Virtual de Química, 3: 816-876.
• Brasil. 2017. Manual de Vigilância da Leishmaniose Tegumentar Americana Ministério da Saúde, Secretaria de Vigilância em Saúde, Brasília, 189p. ( (https://bvsms.saude.gov.br/bvs/publicacoes/manual_vigilancia_leishmaniose_tegumentar.pdf ). Accessed on 30 Jun 2021.
  » https://bvsms.saude.gov.br/bvs/publicacoes/manual_vigilancia_leishmaniose_tegumentar.pdf
• Bustamante, C.; Ochoa, R.; Asela, C.; Muskus, C. 2019. Repurposing of known drugs for leishmaniasis treatment using bioinformatic predictions, in vitro validations and pharmacokinetic simulations. Journal of Computer-aided Molecular Design, 33: 845-854.
• Caetano, A.H.D.; Tomiotto-Pellissier, F.; Miranda-Sapla, M.M.; Assolini, J.P.; Panis, C.; Kian, D. et al 2019. Quercetin promotes antipromastigote effect by increasing the ROS production and anti-amastigote by upregulating Nrf2/HO-1 expression, affecting iron availability. Biomedicine & Pharmacotherapy, 113: 108745.
• Chagas, A.F.S.; Porchia, M.; Nascimento, T.P.; Correa, C.N.S.; Brasil, A.M.V.B.; Franco, A.M.R. 2021. In vitro and in vivo activity of a hypotoxic copper(I) complex against dermotropic Leishmania species. Acta Amazonica, 51: 260-269.
• Chan, C.F.; Liu, Z.; Wong, I.L. K.; Zhao, X.; Yang, Z.; Zheng, J. et al 2021. Amine-Linked Flavonoids as Agents Against Cutaneous Leishmaniasis. Antimicrobial Agents and Chemotherapy, 65: e02165-20.
• Comandolli-Wyrepkowski, C.D.; Jensen, B.B.; Grafova, I.; Santos, P.A.; Barros, A.M.C.; Soares, F.V.; Barcellos, J.F.M. et al 2017. Antileishmanial activity of extracts from Libidibia ferrea: development ofin vitro and in vivo tests.Acta Amazonica, 47: 331-340.
• Dea-Ayuela, M.A.; Rama Iniguez, S.; Alunda, J.M.; Bolás-Fernandez, F. 2007. Setting new immunobiological parameters in the hamster model of visceral leishmaniasis for in vivo testing of antileishmanial compounds. Veterinary Research Communications, 31: 703-717.
• DNDi. 2018. Iniciativa Medicamentos para Doenças Negligenciadas. ( (https://dndial.org/doencas/leishmaniose-cutanea#projetos/ ). Accessed on 5 Jan 2022.
  » https://dndial.org/doencas/leishmaniose-cutanea#projetos/
• Franco, A.M.R.; Grafova, I.; Soares, F.V.; Gentile, G.; Wyrepkowski, C.D.C.; Bolson, M.A.; et al 2016. Nanoscaled hydrated antimony (V) oxide as a new approach to first-line antileishmanial drugs. International Journal of Nanomedicine, 11: 6771-6780.
• Gervazone, L.F.; Ozório, G.G.; Amaral, E.E.A. 2018. 2’-Hydroxyflavanone activity in vitro and in vivo against wild-type and antimony-resistant Leishmania amazonensis PloS Neglected Tropical Diseases, 12: e0006930.
• Gomes, M.N.; Muratov, E.N.; Pereira, M.; Peixoto, J.C.; Rosseto, L.P.; Cravo, P.V.; Andrade, C.H.; Neves, B.J. 2017. Chalcone derivatives: promising starting points for drug design. Molecules, 22: 1210. doi.org/10.3390/molecules22081210
  » https://doi.org/10.3390/molecules22081210
• Glans, H.; Dotevall, L.; Sobirk, S.K.; Farnert, A.; Bradley, M. 2018. Cutaneous, mucocutaneous and visceral leishmaniasis in Sweden from 1996-2016: A retrospective study of clinical characteristics, treatments and outcomes. BMC Infectious Diseases, 18: 1-10. doi: 10.1186/s12879-018-3539-1
  » https://doi.org/10.1186/s12879-018-3539-1
• Hager- Theodorides, A.L.; Massouras, T.; Simitzis, P.E.; Moschou, K.; Zoidis, E.; Sfakianaki, E.; Politi, K.; Charismiadou, M.; Golioumytus, M.; Deligeorges, S. 2021. Hesperidin and naringin improve broiler meat fatty acid profile and modulate the expression of genes involved in fatty acid β-oxidation and antioxidant defense in a dose dependent manner. Foods, 10: 739. doi.org/10.3390/foods10040739
  » https://doi.org/10.3390/foods10040739
• Jourdan, J.P.; Bureau, R.; Rochais, C.; Dallemagne, P. 2020. Drug repositioning: a brief overview. Journal of Pharmacy and Pharmacology, 72: 1145-1151.
• Nassif, P.W.; Mello, T.F.P.; Navasconi, T.R.; Mota, C.A.; Demarchi, I.G.; Aristides, S.M.A.; Lonardoni, M.V.C.; Teixeira, J.J.V.; Silveira, T.G.V. 2017. Safety and efficacy of current alternatives in the topicaltreatment of cutaneous leishmaniasis: a systematic review. Parasitology, 144: 995-1004.
• Pandey, P.; Khan, F.; Maurya, P. 2021. Targeting Jab1 using hesperidin (dietary phytocompound) for inducing apoptosis in HeLa cervical cancer cells. Journal of Food Biochemistry, 45: e13800.
• Quintana, E.; Torres, Y.; Alvarez, C.; Rojas, A.; Forero, M. E.; Camacho, M. 2010. Changes in macrophage membrane properties duringearly Leishmania amazonensis infection differ from those observed during established infection and are partially explained by phagocytosis. Experimental Parasitology, 124: 258-264.
• Reis, L.C.; Brito, M.E.F.; Souza, M.A.; Pereira, V.R.A. 2006. Mecanismos imunológicos na resposta celular e humoral na leishmaniose tegumentar americana. Revista de Patologia Tropical, 35: 103-115
• Sampaio, R.N.R.; Takano, G.H.S.; Malacarne, A.C.B.; Pereira, T.R.; Magalhães, A.V. 2003. Ineficácia in vivo da terbinafina em leishmaniose cutânea causada por Leishmania (Leishmania) amazonensis em camundongos C57BL/6. Revista da Sociedade Brasileira de Medicina Tropical, 36: 531-533.
• Servier. 2019. Laboratórios Servier do Brasil Ltda. DAFLON. Comprimidos. Bula. Technical information. Rio de Janeiro. (https://servier.com.br/wp-content/uploads/2019/04/19.04.17_Daflon_Bula_Paciente.pdf).
  » https://servier.com.br/wp-content/uploads/2019/04/19.04.17_Daflon_Bula_Paciente.pdf
• Silva, D.G. 2008. Padronização do cultivo de amastigotas axênicos e intracelulares de Leishmania spp. e análise da atividade leishmanicida de chalconas. Master’s dissertation, Universidade de Santa Catarina, Brazil, 120p. (https://repositorio.ufsc.br/xmlui/handle/123456789/91195).
  » https://repositorio.ufsc.br/xmlui/handle/123456789/91195
• SVS. 2020. Secretaria de Vigilância em Saúde. Ministério da Saúde. Nota Informativa # 13/2020-CGZV/DEIDT/SVS/MS. ( (https://www.gov.br/saude/pt-br/centrais-de-conteudo/publicacoes/estudos-e-notas-informativas/2020/nota-informativa-miltefosina.pdf/view ). Accessed on 10 Sep 2023.
  » https://www.gov.br/saude/pt-br/centrais-de-conteudo/publicacoes/estudos-e-notas-informativas/2020/nota-informativa-miltefosina.pdf/view
• Tabrez, S.; Rahman, F.; Ali, R.; Akand, S.K.; Alaidarous, M.A.; Banawas, S.; Dukhyil, A.A.B.; Rub, A. 2021a. Hesperidin targetsLeishmania donovanisterol C-24 reductase to fight against leishmaniasis. ACS Omega, 6: 8112-8118.
• Tabrez, S.; Rahman, F.; Ali, R.; Alouffi, A.S.; Akand, S.K.; Alsherhi, B.M.; et al 2021b. Cynaroside inhibits Leishmania donovani UDP-galactopyranose mutase and induces reactive oxygen species to exert antileishmanial response. Bioscience Reports, 41: BSR20203857.
• Teles, G.B.C.; Rodrigues, S.M.; Medeiros, M.G.; et al 2016. Phlebotomine sandfly (Diptera: Psychodidae) diversity and their Leishmania DNA in a hot spot of American cutaneous leishmaniasis human cases along the Brazilian border with Peru and Bolívia. Memórias do Instituto Oswaldo Cruz, 111: 423-432.
• WHO. 2021. World Health Organization. Leishmaniasis. ( (https://www.who.int/health-topics/leishmaniasis#tab=tab_1 ). Acessed on 22 Feb 2021.
  » https://www.who.int/health-topics/leishmaniasis#tab=tab_1
• Xue, H.; Li, J.; Xie, H.; Wang, Y. 2018. Review of drug repositioning approaches and resources. International Journal of Biological Sciences, 14: 1232-1244.

• CITE AS:
  Brasil, A.M.V.; Lopes, J.R.F.; Chagas, A.F.S.; Franco, A.M.R. 2024. Therapeutic potential of hesperidin methyl chalcone in the experimental treatment of cutaneous leishmaniasis. Acta Amazonica 54: e54cp22318.

Data availability

The data that support the findings of this study are not publicly available.

Edited by

• ASSOCIATE EDITOR:
  Emiliano Barreto

Publication Dates

• Publication in this collection
  08 Jan 2024
• Date of issue
  Jan-Mar 2024

History

• Received
  29 Nov 2022
• Accepted
  11 Aug 2023