Leishmaniasis is a neglected tropical disease that is endemic in 98 countries and present in 200 territories, and 12 million people are affected worldwide. Visceral leishmaniasis is a form of this disease with a 95% fatality rate in untreated cases, and it is estimated that 30,000 new cases occur annually (Ruiz-Postigo et al., 2021). This disease has a high prevalence in Brazil, where 96% of cases are reported (Pan American Health Organization, 2018).

In endemic areas of visceral leishmaniasis, a rise in Leishmania–HIV coinfection has been observed. Visceral leishmaniasis accelerates HIV replication and progression, and HIV can influence the disease’s epidemiology, clinical manifestations, and course. Additionally, Leishmania–HIV coinfected patients are more difficult to treat, and they do not respond well to standard treatments, facing more frequent and more severe side effects and higher risks of disease recurrence and death (Alvar et al., 2008; van Griensven and Diro, 2012; DNDi, 2022).

Current treatment for visceral leishmaniasis is based on drugs such as pentavalent antimonials, which have been used as first-line agents for many years, and amphotericin B deoxycholate and lipid formulation amphotericin B (Uliana et al., 2017). These drugs are administered intramuscularly, leading to long-established administration, high cost, and high toxicity. Additionally, pentavalent antimonials are becoming increasingly ineffective due to resistance, and liposomal amphotericin B is a high-cost formulation, making this drug prohibitive in several endemic regions (Uliana et al., 2017; Zulfiqar et al., 2017). Miltefosine has emerged as an alternative treatment for visceral leishmaniasis; however, it is expensive and teratogenic, and its use is not licensed worldwide (Rijal et al., 2013). Therefore, it is necessary to search for new alternatives for the treatment of visceral leishmaniasis. In this context, several natural products have been demonstrated to have antileishmanial activities (Gervazoni et al., 2020).

We had previously demonstrated that apigenin, a flavonoid present in common fruits and vegetables, such as parsley, lemons and berries, has antiparasitic activity in vitroin vitro against Leishmania amazonensis-infected macrophages and can be used as an oral treatment in an experimental model of cutaneous leishmaniasis caused by L. amazonensis (Fonseca-Silva et al., 2016; Emiliano and Almeida-Amaral, 2018). In the present study, we investigated the in vitro activity of apigenin against promastigote and intracellular amastigotes of Leishmania infantum, the etiological agent of visceral leishmaniasis in the New World, and its in vivo activity in an experimental model of visceral leishmaniasis caused by L. infantum.

To evaluate the in vitro effect of apigenin, promastigote forms and intracellular amastigote forms of L. infantum were used. Promastigotes were incubated in the absence or presence of different concentrations of apigenin (12-96 µM) for 72 h ( Figure 1 ). Apigenin inhibited the cellular proliferation of L. infantum in a concentration-dependent manner, reaching 94.6% inhibition at the highest concentration (96 µM) with an IC₅₀ value of 29.9 µM.

To determine the effect of apigenin on L. infantum intracellular amastigotes, peritoneal BALB/c mouse macrophages were infected with promastigotes of L. infantum for 5 h and then incubated with increasing concentrations of apigenin (3-24 μM) for 72 h. Apigenin was able to reduce the infection index in a concentration-dependent manner ( Figure 2 ). The IC₅₀ was 2.3 µM, reaching 88% inhibition at the highest concentration (24 µM). The reported CC₅₀ value of apigenin is 78.7 µM (Fonseca-Silva et al., 2016), and the selectivity index (SI) was calculated to be 34.3.

According to the observed in vitro effect of apigenin, the efficacy of this molecule in vivo was evaluated using a murine model of visceral leishmaniasis using two therapeutic schemes. The first was short-term, where L. infantum-infected BALB/c mice were treated for 5 days beginning at 7 days post-infection and euthanized at 2 days post-treatment (Day 14). In the long-term therapeutic scheme, L. infantum-infected BALB/c mice were also treated for 5 days beginning at 7 days post-infection, but euthanization occurred at 18 days post-treatment (Day 30) (Le Fichoux et al., 1998).

As shown in Figure 3 , during the short-term treatment, apigenin orally administered to L. infantum-infected BALB/c mice reduced the liver-parasite load, reaching 99.7% inhibition. However, we did not observe any significant differences between mice treated with apigenin and those treated with meglumine antimoniate.

Furthermore, serological toxicology parameters, such as total levels of albumin, creatinine kinase, urea, alkaline phosphatase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol, iron, calcium, sodium, and potassium ( Supplementary Table 1 ), and several hematological parameters ( Supplementary Table 2 ) were evaluated, and no significant alterations were observed, suggesting a lack of toxicity.

In the long-term therapeutic scheme ( Figure 4 ), oral administration of apigenin to L. infantum-infected BALB/c mice resulted in a 94% reduction in the liver parasite load. Furthermore, differences between the infected mice treated with apigenin and meglumine antimoniate were observed in terms of parasite load, and meglumine antimoniate reduced the liver parasite load by only 55%.

Current visceral leishmaniasis chemotherapy has presented many problems, such as several collateral effects, increased ineffectiveness due to resistance, and high cost making these drugs unaffordable in several endemic regions. Therefore, more research is necessary to improve this drug arsenal, giving priority to compounds with greater efficacy, reduced toxicity, and better affordability. Natural products have become an interesting alternative, and flavonoids have been studied as promising compounds for leishmaniasis treatment (Fonseca-Silva et al., 2011; Inacio et al., 2012; Fonseca-Silva et al., 2013; Inacio et al., 2013; Ndjonka et al., 2013; Inacio et al., 2014; Fonseca-Silva et al., 2015; Fonseca-Silva et al., 2016; Emiliano and Almeida-Amaral, 2018; Gervazoni et al., 2018; Inacio et al., 2019; Gervazoni et al., 2020). Apigenin has demonstrated a key role in the treatment of some health issues, such as diabetes, amnesia and Alzheimer’s disease, depression, insomnia, and cancer (Salehi et al., 2019). Moreover, the activity of apigenin has been described against L. donovani, T. brucei rhodesiense, T. cruzi, Encephalitozoon intestinalis and Cryptosporidium parvum (Mead and McNair, 2006; Tasdemir et al., 2006).

Cellular proliferation of the promastigote forms of L. infantum in the presence of apigenin was inhibited in a concentration-dependent manner, presenting an IC₅₀ of 29.9 µM. A similar effect of apigenin was observed in the promastigote forms of L. amazonensis, which showed an IC₅₀ value of 23.7 µM (Fonseca-Silva et al., 2015).

Some conditions for the eligibility of a new drug for the treatment of visceral leishmaniasis have been described. The new drug should have an IC₅₀ less than 10 μM against intracellular amastigotes; be, at a minimum, tenfold more active against intracellular amastigotes than against mammalian cells (SI ≥ 10); be active in vivo; and cause a reduction in liver parasite load more than 70% in a relevant small animal model after at most 5 doses at 50 mg/kg, preferably when administered by the oral route once or twice per day (Pink et al., 2005; Katsuno et al., 2015).

In the present study, we demonstrated that apigenin was active against intracellular amastigotes, with an IC₅₀ of 2.3 µM and a selectivity index of 34.3. We also observed that the treatment of BALB/c mice infected with L. infantum with apigenin (1 mg/kg twice a day, reaching a maximum daily dose of 2 mg/kg/day, orally administered) was capable of reducing the liver parasite load by greater than 70% in both therapeutic schemes (short-term and long-term). It is important to point out that the treatment of apigenin did not compromise the overall health of the infected mice, satisfying all the criteria described above. A similar effect was observed with (-)-epigallocatechin 3-O-gallate, an abundant flavonoid constituent of green tea, that had an IC₅₀ value of 2.6 µM for intracellular amastigotes and demonstrated a reduction of the liver parasite load, reaching 98.7% inhibition at 50 mg/kg/day (Inacio et al., 2019).

Taken together, the results indicate apigenin as a new compound for the treatment of visceral leishmaniasis and support further studies to determine the ideal therapeutic and optimal drug dose regimen.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by L-11/2017 A2.

Conceptualization, YE and EA-A; Methodology, YE and EA-A; Validation, YE and EA-A; Formal analysis, YE; Investigation, YE and EA-A; Data curation, YE and EA-A; Writing—original draft preparation, YE and EA-A; Writing—review and editing, EA-A; Supervision, EA-A. All authors contributed to the article and approved the submitted version.