Antileishmanial Activities of Medicinal Herbs and Phytochemicals In Vitro and In Vivo: An Update for the Years 2015 to 2021

Leishmaniasis is one of the most neglected tropical diseases that present areal public health problems worldwide. Chemotherapy has several limitations such as toxic side effects, high costs, frequent relapses, the development of resistance, and the requirement for long-term treatment. Effective vaccines or drugs to prevent or cure the disease are not available yet. Therefore, it is important to dissect antileishmanial molecules that present selective efficacy and tolerable safety. Several studies revealed the antileishmanial activity of medicinal plants. Several organic extracts/essential oils and isolated natural compounds have been tested for their antileishmanial activities. Therefore, the aim of this review is to update and summarize the investigations that have been undertaken on the antileishmanial activity of medicinal plants and natural compounds derived, rom plants from January 2015 to December 2021. In this review, 94 plant species distributed in 39 families have been identified with antileishmanial activities. The leaves were the most commonly used plant part (49.5%) followed by stem bark, root, and whole plant (21.9%, 6.6%, and 5.4%, respectively). Other plant parts contributed less (<5%). The activity was reported against amastigotes and/or promastigotes of different species (L. infantum, L. tropica, L. major, L. amazonensis, L. aethiopica, L. donovani, L. braziliensis, L. panamensis, L. guyanensis, and L. mexicana). Most studies (84.2%) were carried out in vitro, and the others (15.8%) were performed in vivo. The IC50 values of 103 plant extracts determined in vitro were in a range of 0.88 µg/mL (polar fraction of dichloromethane extract of Boswellia serrata) to 98 µg/mL (petroleum ether extract of Murraya koenigii). Among the 15 plant extracts studied in vivo, the hydroalcoholic leaf extract of Solanum havanense reduced parasites by 93.6% in cutaneous leishmaniasis. Voacamine extracted from Tabernaemontana divaricata reduced hepatic parasitism by ≈30 times and splenic parasitism by ≈15 times in visceral leishmaniasis. Regarding cytotoxicity, 32.4% of the tested plant extracts against various Leishmania species have a selectivity index higher than 10. For isolated compounds, 49 natural compounds have been reported with anti-Leishmania activities against amastigotes and/or promastigotes of different species (L. infantum, L. major, L. amazonensis, L. donovani and L. braziliensis). The IC50 values were in a range of 0.2 µg/mL (colchicoside against promastigotes of L. major) to 42.4 µg/mL (dehydrodieuginol against promastigotes of L. amazonensis). In conclusion, there are numerous medicinal plants and natural compounds with strong effects (IC50 < 100 µg/mL) against different Leishmania species under in vitro and in vivo conditions with good selectivity indices (SI > 10). These plants and compounds may be promising sources for the development of new drugs against leishmaniasis and should be investigated in randomized clinical trials.

Latest developments in the prevention and treatment regarding a permanent solution for leishmaniasis in terms of successful human vaccination is still a major challenge. However, there are different vaccinations currently being tested in mouse models. One of them uses "killed but metabolically active" parasites to induce host immune system reaction. Using salivary peptides of the sandfly holds the potential to be used as a vaccine component. However, the complex immune response makes it a challenge [6]. Macrophage-targeted drug delivery systems are another novel approach to directly affect Leishmania parasites that live in the macrophages. As getting into macrophages is a challenge, liposomes, microspheres, nanoparticles, and carbon nanotubes are some of the various drug carriers that are studied to target macrophages. In addition, the use of specific receptors expressed by macrophages to actively deliver a drug is also used [7].
The current treatment by chemical drugs has several limitations such as toxic side effects, high costs, frequent relapses, the development of resistance, and the requirement for long-term treatment [8,9]. Thus, investments in novel drug development against this parasitic disease may be a risky affair. Medicinal plants are centuries-old sources in the various traditional herbal medicine systems of the world. For instance, their importance lies in the fact that the WHO concludes that about 80% of the world's population relies on them for primary health care [10]. Moreover, 25 to 50% of the pharmacopeias worldwide contain plant products and drugs derived from natural products [11]. Therefore, current research approaches for the treatment of leishmaniasis should largely consider medicinal plants as an important area of search.
The aim of this review is to update and summarize the investigations that have been undertaken on the antileishmanial activity of medicinal plants and natural compounds derived from plants from January 2015 to December 2021. Table 2, 92 plant species distributed in 39 families have been identified with anti-Leishmania activities. The family Fabaceae accounted for the highest percentage (9.7%) followed by Asteraceae (7.6%). Lamiaceae and Solanaceae account for 6.5% each. Table 2. Botanical characteristics of the medicinal herbs in the present study.

No
Family Name Scientific Name Part Used

Spondias mombin Leaves
The leaves were the most commonly used plant part as compared to other parts (49.5%) followed by stem bark, roots, and whole plant (21.9%, 6.6%, and 5.4%, respectively). Aerial parts and fruits accounted for 4.5% each. Other plant parts (flowers, seeds, resins, branches, and kernels) contributed less (<4%) (Figure 1). The leaves were the most commonly used plant part as compared to other parts (49.5%) followed by stem bark, roots, and whole plant (21.9%, 6.6%, and 5.4%, respectively). Aerial parts and fruits accounted for 4.5% each. Other plant parts (flowers, seeds, resins, branches, and kernels) contributed less (<4%) (Figure 1). With respect to the test methods, 84.2% of studies were carried in vitro, while 15.8% of them were performed using in vivo assays (Tables 3 and 4). For in vitro assay, 80 medicinal plants were screened in vitro for antileishmanial activities against different Leishmania species (L. infantum, L. tropica, L. major, L. amazonensis, L. aethiopica, L. donovani, L. braziliensis, L. panamensis, L. guyanensis, and L. mexicana) and life cycle forms (amastigotes and/or promastigotes). The IC50 value of 103 plant extracts/essential oils determined in vitro was in a range of 0.88 μg/mL (polar fraction of dichloromethane extract of Boswellia serrata) to 98 μg/mL (petroleum ether extract of Murraya koenigii) (  With respect to the test methods, 84.2% of studies were carried in vitro, while 15.8% of them were performed using in vivo assays (Tables 3 and 4). For in vitro assay, 80 medicinal plants were screened in vitro for antileishmanial activities against different Leishmania species (L. infantum, L. tropica, L. major, L. amazonensis, L. aethiopica, L. donovani, L. braziliensis, L. panamensis, L. guyanensis, and L. mexicana) and life cycle forms (amastigotes and/or promastigotes). The IC 50 value of 103 plant extracts/essential oils determined in vitro was in a range of 0.88 µg/mL (polar fraction of dichloromethane extract of Boswellia serrata) to 98 µg/mL (petroleum ether extract of Murraya koenigii) ( Table 3). B. serrata (resins), R. officinalis (leaves), A. riparia (fruits), M. pulegium (leaves) as extracts had strong anti-Leishmania activity (0.88, 1.2, 1.3, and 1.3 µg/mL, respectively).

Solanaceae
For in vivo assay, among the 15 medicinal plants studied in vivo, the highest activity against cutaneous leishmaniasis was exhibited by the hydroalcoholic leaf extract of Solanum havanense, which reduced parasites by 93.6%, and the highest activity against visceral leishmaniasis was shown by the voacamine compound extracted from Tabernaemontana divaricata, which reduced the hepatic and splenic parasitism by ≈30 times and ≈15 times, respectively (Table 4). For cytotoxic activity, 32.4% of tested plant extracts have good cytotoxic activity with a selectivity index of SI > 10. (Table 5).
For isolated compounds, 49 natural compounds have been identified with anti-Leishmania activities against amastigotes and/or promastigotes of different species (L. infantum, L. major, L. amazonensis, L. donovani and L. braziliensis). The IC 50 values were in the range of 0.2 µg/mL (colchicoside against promastigotes of L. major) to 42.4 µg/mL (dehydrodieuginol against promastigotes of L. amazonensis) ( Table 6).               Numerous natural compounds were isolated from different parts of the plants that were used in traditional medicine to treat leishmaniasis [67]. These compounds act against Leishmania by various mechanisms including the disintegration of cytoplasmic membranes, electron flow disturbances, active transport of crucial substances, coagulation of the cell contents, and destabilization of proton motive forces [68]. For example: • Some medicinal plants are enriched with essential oils composed of different hydrophobic molecules which can diffuse easily across cell membranes and consequently gain access to intracellular targets [67,69]. They may also act on ATPases and other proteins located in cytoplasmic membranes that are surrounded by lipid molecules. They can also cause a distortion of lipid-protein interactions in hydrophobic parts of the proteins, or they can interact with the enzymes involved in the synthesis of structural sections.

•
The diversity of terpenoids increases their biological activity spectrum, including several Leishmania species [70]. Terpenes can easily penetrate the lipid bilayer of the cell membrane and produce changes in the integrity of cell structure and the mitochondrial membrane of Leishmania parasites [67]. For example, Artemisinin induced apoptosis, depolarization of the mitochondrial membrane potential, and DNA fragmentation [71,72]. Ursolic acid induce programmed cell death independent of caspase 3/7 but dependent on mitochondria. The compound reduced the lesion size and parasite load of cutaneous leishmaniasis in vivo [70]. (−)-α-Bisabolol induced phosphatidylserine externalization and caused cytoplasmic membrane damage, both of which are apoptosis indicators. The compound also decreased ATP levels and disrupted the mitochondrial membrane potential [73]. • Plants enriched with antioxidant compounds such as flavonoids may act by initiating morphological changes and causing a loss of cellular integrity, leading to cell cycle arrest in the G1 phase [59]. They also may act by damaging the mitochondria of the parasites [67]. For example, apigenin increased intracellular reactive oxygen species (ROS) and the number of double-membrane vesicles as well as myelin-like membrane inclusions, which are characteristics of the autophagic pathway. Furthermore, the fusion between autophagosome-like structures and parasitophorous vacuoles was observed [65]. Epigallocatechin 3-O-gallate (EGCG) has increased ROS levels, which decreased the mitochondrial membrane potential and the ATP levels [58].

•
The diversity of structures within the coumarin group enables them to exhibit many biological activities, including anti-Leishmania activity. It represents a promising natural compound that can act on two fronts: as a treatment for leishmaniasis (able to induce mitochondrial membrane damage and changes in ultrastructure [74] and as a tool to control Leishmania vectors (might block the transmission of leishmaniasis since they decrease parasite loads [27]. • Many alkaloids have been described as having biological activities against trypanosomatids, such as Leishmania spp. For example, heterocyclic steroids (solamargine and solasonine) induced different immunochemical pathways in macrophages and dendritic cells. Additionally, they were capable of enhancing the expression levels of transcription factors, such as NFκB/AP-1 [43]. In addition, isoquinoline alkaloid (berberine) has leishmanicidal activity through a reduction in the viability of promastigotes and the generation of ROS in these cells. It also increased the levels of mitochondrial superoxide and induced the depolarization of mitochondrial transmembrane potential [53].

Study Design and Setting
In order to perform this review, the following aspects were addressed: identification and selection of the theme of the research question, establishment of criteria for selection of the sampling, the definition of information to be extracted from selected studies, assessment of the studies included in the integrative review, and final explanation of the results.

Search Strategies
The databases used for this article were PubMed, Google Scholar, Web of Science, Research Gate, SCOPUS, and Scientific Electronic Library Online (SciELO) using the keywords: neglected tropical disease, Leishmania species, anti-Leishmania activity, natural product, medicinal plants, and promastigote form. We used the search terms separately and in combination with the Boolean operators "OR" or "AND".

Inclusion and Exclusion Criteria
The initial total articles (1374) were adjusted for the restriction in the year of publication (from 1 January 2015 to 31 December 2021) (806), duplicates (273), articles that were not available in full (67) and articles in other languages (4). After a review of their titles and abstracts, some articles were discarded, since the anti-leishmanial activity (IC 50 ) values were higher than 100 µg/mL (134), and they tested extract/natural compounds obtained through other natural sources (algae, fungi, etc.) (11). The full texts of the remaining articles were reviewed in detail. However, further articles were discarded after the full text had been reviewed (18) since they did not address much of the required information. Finally, 61 articles were evaluated as valuable to reach the goals of this review. The methodological validity of all 61 studies was proven prior to inclusion in the review by undertaking a critical appraisal using a standardized instrument [75].

Data Extraction and Analysis
The data extraction protocol included the scientific and family names, parts of the plant used, most active extract/ essential oil employed in the experiment, name of natural compound, Leishmania species and form, IC 50 values, potential groups/compounds responsible for activity, clinical form of leishmaniasis, route, the dose of administration and scheme of treatment, the efficacy of the treatments in the experiment, cytotoxic activity, selectivity index, the authors, and year of publication. In the results analysis, an active extract/compound was considered if the IC50 value was less than or equal to 10 µg/mL against the promastigote or amastigote forms. Moderate activity was defined if the IC50 was greater than 10 and less than 50 µg/mL and weakly active if the IC50 value was greater than 50 µg/mL and less than 100 µg/mL.

Conclusions and Perspectives
Leishmaniasis threatens about 350 million people around the world and continues to represent a menace on a global scale. Without a doubt, it requires utmost attention due to the lack of vaccines for the prevention and reported resistance against available chemical drugs for treatment. The intolerably high incidence of millions of new cases of leishmaniasis per year worldwide and deficiencies in current treatment point to an urgent need for new medications.
As a means to facilitate the accessibility of information, this review updates and summarizes recent results on medicinal plants and natural compounds against different Leishmania species. The plants presented here have demonstrated a diverse range of activities against different forms of leishmaniasis with some showing high activities that could be reasonable starting points for the further development of effective and affordable novel drugs.
However, it was also evident that the majority of experiments were performed with the promastigote form. We believe that these studies are undoubtedly important because promastigotes are infectious to man and other animals. However, it is urgent that future studies should be conducted to find compounds with anti-amastigote activity too, since the morbimortality associated with Leishmania is caused by this form.
It Is pleasing that more and more investigations report on the anti-Leishmania activity in vivo and more studies are needed in this respect, increasing the number of potential candidate compounds for further drug development. In vitro studies are valuable for the screening of extracts and isolated compounds as well as for investigations of the cellular and molecular modes of action. Since many natural compounds are rapidly metabolized in the human body by liver enzymes and gastrointestinal microflora, animal experiments are indispensable to identify candidates with sufficient half-life times in vivo and anti-Leishmania activities in concentration ranges that are reachable in the human blood. However, in the literature inspected by us, only four plants and two natural compounds have been investigated both in vitro and in vivo, i.e., Prosopis juliflora [34], Ziziphus spinachristi [37], Piper pseudoarboreum [33], and Croton caudatus [76] as well as epigallocatechin 3-O-gallate [58] and apigenin [65], respectively. More investigations are required to allow a direct comparison of in vitro and in vivo data.
Further down this line of argumentation, standardized extracts and/or isolated phytochemicals need to be tested in randomized clinical trials. Without convincing clinical evidence on safety and efficacy, preparations from traditional medicine will hardly reach considerable recognition in the medical world.