An Update on Natural Antileishmanial Treatment Options from Plants, Fungi and Algae

Efficient drugs for the treatment of leishmaniasis, which is classified as a neglected tropical disease, are sought for. This review covers potential drug candidates from natural plant, fungus and algae sources, which were described over the last six years. The identification of these natural antileishmanials often based on the knowledge of traditional medicines. Crucial insights into the activities of these natural remedies against Leishmania parasites and against infections caused by these parasites in laboratory animals or patients are provided and compared with selected former active examples published more than six years ago. In addition, immuno‐modulatory natural antileishmanials and recent developments on combination therapies including natural products and approved antileishmanials are discussed. The described natural products revealed promising data warranting further efforts on the discovery and development of new antileishmanials based on patterns from nature.


Introduction
Neglected tropical diseases (NTDs) pose a constant threat to world health and comprise a number of infectious diseases, which affect and endanger billions of mainly poor people worldwide. However, the development of accurate treatment options for these poor populations is still not secured. [1] Leishmaniasis is an NTD, which, in its various forms, is endemic to vast areas from South Asia via the Middle East and Africa to South and Central America. [1] It is clinically subdivided into visceral leishmaniasis (VL, caused by L. infantum and L. donovani), cutaneous leishmaniasis (CL, caused by L. infantum, L. major, L. tropica, L. mexicana, L. amazonensis, and L. braziliensis), and mucocutaneous leishmaniasis (MCL, caused by L. braziliensis and L. donovani). VL has a considerable mortality potential while CL is the predominant form of leishmaniasis with up to 1 million new cases annually. [2,3] Current treatments of leishmaniasis patients include antimoni-als, amphotericin B, miltefosine, paromomycin, and pentamidine, but the systemic toxicity of widely applied pentavalent antimonials and the appearance of drug-resistance necessitate the search for new potent drugs against leishmaniasis. [3] Leishmania parasites appear as extracellular motile promastigotes and intracellular/intra-macrophage amastigotes. Both forms are generally applied for antileishmanial research, however, there are differences, which affect the validity of the drug testing experiments. There is general strong support of using the more laborious in vitro tests with intracellular amastigotes for antileishmanial drug discovery because they cover hostmediated mechanisms, too. In contrast, cell-free assays with promastigotes are simpler and more proper for automation but can lead to the identification of false positives (i. e., compounds which are active against short-lived promastigotes but inactive against cellprotected amastigotes) and are mainly recommended share of new investigational drugs. [6] In wide parts of the Asian continent, traditional folk medicine such as Traditional Chinese Medicine, Kampo, Ayurveda, Unani, and traditional Greco-Arab and Islamic Medicine, among others, are valuable sources for the discovery of natural drugs. [7,8] But folk medicine is not confined to Asia and is also practiced by native populations in wide parts of Africa and America. For instance, various Bolivian medicinal plants can be considered as suitable treatment options for the management of Leishmania infections. [9] The role of natural products in drug discovery is axial, ancient and universal. According to the WHO, over 70 % of the world population still relies on herbal remedies in terms of healthcare. [10] The antileishmanial potential of secondary metabolites and extracts of plants was reviewed. [11][12][13] Natural products from plants such as terpenes, alkaloids and polyphenols have shown distinct activities against leishmaniasis. [14 -16] The anti-leishmanial activities of the natural phenol curcumin (1) and of naphthoquinones such as plumbagin (2, ex. Plumbago sp.) and 2hydroxy-1,4-naphthoquinone (lawsone, ex. Lawsonia inermis) including the semi-synthetic derivatives of the latter were recently described. [17 -20] The sources of natural products used against various ailments are not limited to plants. Major groups of organisms, whose extracts and isolates were subjected to antiparasitic studies, include algae, cyanobacteria, and fungi. [21 -31] This review aims at a concise description of nature-derived medications and active principles for the treatment of various leishmaniasis forms, which emerged over the last six years. It is focused on plants, algae and fungi samples with considerable antileishmanial activities good enough to be considered as possible treatment options in the future. Further sub-sections include immuno-modulatory natural products, since such immunomodulators experienced a rising interest over the last years, and combination therapies of natural products with currently applied antileishmanials.
Summing up, the described antileishmanial natural compound classes comprise terpenes, quinones, alkaloids, and various aromatic compounds such as phenols and flavones. Natural products such as linalool, nerolidol, curcumin, and plumbagin are structurally simple and in parts commercially available, which should enable the future semi-synthesis of derivatives of these compounds with improved antileishmanial activities. Flavones such as luteolin are also easily and commercially available and might serve as starting points for the semi-syntheses of new antileishmanial flavone derivatives. Table 1 summarizes the plants and their isolates, which were used for the treatment of various types of leishmaniasis. More plant derived products with immune-modulatory properties are mentioned separately for a better clarity.
In addition to plants, other natural sources such as algae and fungi provide compounds with antileishmanial activities. Various hexane/ethyl acetate eluent fractions were obtained from the Antarctic Iridaea cordata red alga by chromatographic purification and two polar fractions (i. e., fractions obtained with an eluent with a high content of ethyl acetate) showed activity against L. amazonensis promastigotes (IC 50 = 17.4 μg/mL for hexane: AcOEt = 1 : 4 fraction, 24.3 μg/ mL for hexane: AcOEt = 2 : 3 fraction) and amastigotes (IC 50 = 12.4 μg/mL for hexane: AcOEt = 1 : 4 fraction, 4.0 μg/mL for hexane: AcOEt = 2 : 3 fraction). [23] The highest activity was observed against amastigotes for the hexane: AcOEt = 2 : 3 fraction and further efforts to purify the active principle(s) of this fraction appear to be promising. Iberian Cystosteira macroalgae were investigated for antileishmanial activities and the hexane extracts of C. baccata and C. barbata displayed reasonable activities against L. infantum amastigotes (IC 50 = 5.1 μg/mL and 6.8 μg/mL. [53] Carotenoids, ste-roids, meroterpenoids, fatty acids and triacylglycerols were identified as components of the Cystosteira extracts, which warrant further exploration as possible antileishmanial drugs. Various fungi were tested against Leishmania parasites. Water soluble polysaccharides were isolated from the Indian wild mushrooms Termitomyces eurhizus (IC 50 = 100 μg/mL) and Russula laurocerasi (IC 50 = 86.9 μg/mL), which were moderately active against L. donovani amastigotes. [25] Ethanol and ethyl acetate extracts of the mushroom Grifola frondosa were also moderately active against L. donovani promastigotes (IC 50 = 93.9 μg/mL for EtOH extract and 412.5 μg/mL for AcOEt extract). [28] A fungal extract library was also established for new antileishmanial compounds. In this way, the new bisabolane sesquiterpene lactone derivative HD871-1 (18) was discovered with considerable activity against L. mexicana amastigotes (IC 50 = 3.73 μM). [54] The activity of 16 matches with the activities of other fungal sesquiterpenes such as hypnophilin (19, isolated from the mushroom Lentinus strigosus), which exhibited distinct activity against amastigotes of L. amazonensis (IC 50 = 2.16 μg/mL, 8.7 μM). [29] A direct comparison is difficult since different Leishmania strains were applied for testing. A higher activity than those of 18 and 19 against amastigotes of L. donovani was observed for fungal quinones of the endophytic fungus Edenia sp. such as 20 (palmarumycin CP18, IC 50 = 0.62 μM, SI = 245). [26] Nevertheless, compound 18 is clearly of interest as a potential new antileishmanial drug candidate, which might be successfully optimized to new analogs with improved antileishmanial activities by (semi-)synthetic methods in the future. Harzialactone a (21), which was recently isolated from the marine-derived fungus Paecilomyces sp 7 A22, also shows a lactone scaffold. But its antileishmanial activities were only moderate against L. amazonensis promastigotes (IC 50 = 5.25 μg/ mL, 27.3 μM) or weak against L. amazonensis amastigotes (IC 50 = 18.18 μg/mL, 94.6 μM). [55] Table 2 and Figure 2 summarize the outcome of the studies with algae and fungal sources for the identification of antileishmanial constituents.

Synergy Effects of Natural Extracts, Oils and Isolates in Combination with Approved Drugs
The promising effects of natural products in combination with currently approved antileishmanial drugs are briefly described in this section. For instance, the combination of natural products with antimonials can lead to synergy effects and improved treatment outcomes. Meglumine antimoniate in combination with the well-known plant spices capsaicin (29, ex. Capsicum sp., Solanaceae) and piperine (5, Piperaceae, see Figure 1) led to enhanced antileishmanial in vitro activities of both natural compounds against L. infantum promastigotes and amastigotes. [69] In addition, a clinical study with patients suffering from CL using glucantime plus ozonated olive oil exhibited synergy effects leading to distinct lesion size  Figure 1).
Chem. Biodiversity 2022, 19, e202100542 reductions. [70] Such combination strategies can also be suitable options in order to reduce the systemic toxicity of antimonials in case that lower effective antimonial doses can be administered.
Although very efficient, amphotericin B is an expensive antileishmanial drug having side-effects and so synergy effects of natural products with amphotericin B were studied in order to find ways to reduce the necessary amphotericin B doses for leishmaniasis treatments. A study of the ethanolic leaf extract of the African medicinal plant Moringa oleifera (Moringaceae) showed its activity against L. major promastigotes (IC 50 = 6.87 μg/mL) and amastigotes (IC 50 = 9.31 μg/ mL) and revealed a considerable antileishmanial synergy effect in combination with amphotericin B (fractional inhibitory concentration/FIC = 0.375). The active principles of M. oleifera leaf extract were identified as resorcinol (30, IC 50 = 3.79 μg/mL, 34.4 μM), luteolin 7-O-glucoside (31, IC 50 = 10.7 μg/mL, 23.9 μM) and syringic acid (32, IC 50 = 13.4 μg/mL, 67.6 μM). [71] Already two decades ago, the aglycone luteolin (isolated from Vitex negundo, Lamiaceae) and its structurally related flavonoid quercetin (isolated from Fagopyrum esculentum, Polygonaceae) were shown to reduce splenic parasite load in hamsters infected with L. donovani by 80 % (luteolin, 3.5 mg/kg) and 90 % (quercetin, 14 mg/kg) while luteolin remained non-toxic to human cells. [72] Various propolis extracts, oils and compounds showed distinct in vitro and in vivo activities against Leishmania strains and infections. [73] The essential oil of Tunisian propolis, which was made by bees (Apis mellifera) in a region where various Citrus plants (Rutaceae) grow, exhibited strong activities against L. major and L. infantum promastigotes (IC 50 = 5.3 μg/mL and 3.7 μg/mL) and amastigotes (IC 50 = 7.4 μg/mL and 5.0 μg/mL), it activated macrophages by NO formation, and it displayed synergy effects when combined with amphotericin B (FIC = 0.37). [74] Cadambine acid (33) isolated from the Naucleas diderichii (Rubiaceae) medicinal plant growing in West and Central Africa was active against and selective for L. infantum amastigotes (IC 50 = 1.2 μM, SI > 209) by induction of NO formation, and it acted synergistically in concert with amphotericin B. [75] Flavolignans were isolated from milk thistle (Silybum marianum, Asteraceae), and although dehydroisosilybin A (34) showed only low activity against L. donovani and L. infantum promastigotes (IC 50 = 90.2 μM), it exhibited a moderate synergy effect together with amphotericin B, which made it possible to reduce the applied dose of dehydroisosilybin A by a factor of more than 4 and the dose of amphotericin B by a factor of 2. [76] Taken together, there is a well-equipped arsenal of natural products, which were successfully combined with approved antileishmanial drugs such as antimonials and amphotericin B. Quite a few of them such as piperidine, capsaicin, resorcinol, syringic acid, luteolin, olive oil, and propolis are easily available and, thus, can be suitable additives for currently applied treatments in order to reduce costs and side-effects/ toxicities. Structures of natural products with synergy effects when combined with clinically approved drugs are shown in Figure 4.

Conclusions
This review underlines the great potential of natural products as valuable drug candidates for the treat-  Figure 1). ment of leishmaniasis. Nature can provide many possible treatment options for various forms of leishmaniasis. This review does not include bacterial sources, yet, a considerable number of antileishmanial plant, fungal and algae products. These natural sources are already being applied by traditional healers in tropical and sub-tropical regions as treatments for various infectious diseases. A good deal of the described extracts and natural products was derived from plants. Medicinal plant products can enhance the effects of conventional antileishmanials such as antimonials and amphotericin B, which can decrease treatment costs and improve the quality of life of patients due to reduced side-effects. Various products of algae and fungi also revealed promising antileishmanial activities not inferior to nor less promising than those effects of the plant-based products. Several extracts and natural products showed excellent in vitro and/or in vivo antileishmanial activities, which warrant further in-depth studies. The immuno-modulating properties of certain compounds and fractions are particularly interesting since they have the potential to lead to an immunization against Leishmania infection and, thus, they can also be of relevance for other human diseases including infectious diseases such as viral infections.
This review is intended to show the potential of nature-derived antileishmanials and to encourage readers either to start or to continue research in the field of nature-inspired drugs against NTDs such as leishmaniasis. These efforts can comprise the exploitation of currently known plants, fungi, and algae products with known antileishmanial effects, as well as the identification of new natural sources with antileishmanial activities in order to develop cost-effective and easily accessible therapies, which can add to or even replace current standard therapies for leishmaniasis diseases. In addition, chemists can provide synthetic approaches to the described highly active natural products in order to achieve an easier access to these molecules and to generate new (semi-)synthetic derivatives with improved biological properties.