**4. Phytocompound nanoformulations and antileishmanial activity**

In the last decades, advances in nanoscience have enabled the development of nano-range materials approved for therapeutic use or in clinical development stage. In fact, many encapsulation matrices have been approved by the Food and Drug Administration (FDA) for use in humans, i.e., a wide range of naturally or chemically modified cyclodextrins that are extensively used in medicine and food fields [60, 61]. Similarly, vesicular systems such as liposomes, niosomes, nanoparticles, and microspheres are very useful and have many advantages in delivering drugs of natural origin, representing a promising approach for the treatment of several diseases, including leishmaniasis. Nanoformulations prepared with compounds from medicinal plants and their antileishmanial activity are summarized in Table 2. Liposomes, niosomes, and nanoparticles are the main formulations investigated.

Saponins (liposomes) and alkaloids (nanoparticles) are among the main plant constituents used to prepare nanoformulations. Liposomes are mainly formed by a mixture of phosphatidyl choline (PC), cholesterol (Chol), and phosphatidic acid (PA), usually in a 7:4:1 molar ratio [55, 38], whereas nanoparticle formulations have polylactide (PLA) as their main ingredient, providing greater stability, biocompatibility, and an efficient delivery system compared to liposomal structures [50, 57]. The antileishmanial properties of each nanoformulation are briefly cited below.

Flavonoids are among the most common phenolic compounds found in the human diet and a variety of members of this family has been described as bioactive agents. Significant antiprotozoal activity of flavonoids has been reported against *Trypanosoma* and *Leishmania* species. Quercetin (Fig. 1) is a widely studied food-derived flavonoid with several biological effects, including antioxidant, antihypertensive, anti-inflammatory, and antiprotozoal activities. Quercetin inhibits parasite arginase activity [62,63] and induces the production of superoxide anion, hydrogen peroxide, and other reactive oxygen species (ROS) by infected cells. Thus, ROS generation induced by quercetin could be crucial for maximal antiparasitic activity, because ROS are naturally generated by macrophages as a mechanism to kill intra‐ cellular parasites such as *Leishmania* [64, 65]. In fact, liposomal, niosomal, microspherulated, and nanocapsulated quercetin formulations have been tested to evaluate the best drug delivery system. In hamster models of *L. donovani* infection, all quercetin vesicular formulations reduced the parasite load compared to the free form of the drug. Nanocapsulated quercetin is more effective than non-capsulated quercetin in the control of leishmaniasis (87% reduction in spleen parasite burden) and its pronounced activity may be related to vesicular composition and size. Moreover, drug efficacy may be inversely correlated to the size of vesicular forms [39]. Similarly, the incorporation of terpenoids into nanoparticle carriers has also shown promising results. The search for active molecules to treat leishmaniasis is very laborious, because most molecules have low solubility. The incorporation of andrographolide (Fig. 2), a diterpenoid extracted from the herbaceous species *Andrographis paniculata* (Acanthaceae), with different poly (d,l-lactide-co-glycolide) (PLGA) nanoformulations enhanced the antileishmanial activity of andrographolide against axenic and intracellular amastigote forms of *L. donovani*. Among the formulations tested, the 175 nm andrographolide-loaded nanoparticles exhibited the best antileishmanial activity (IC50 = 36 and 28µM for axenic and intracellular amastigotes, respectively) [66]. *Andrographis paniculata* can be considered an interesting source of antileish‐ manial agents. While 14-deoxy-11-oxoandrographolide (Fig. 2), an andrographolide-derived diterpenoid, reduced spleen parasite load in hamster models of *L. donovani* infection in 39%, liposomal, niosomal, and microspherulated formulations of this substance suppressed spleen parasite load by 78, 91, and 59%, respectively. In addition, the toxicity of 14-deoxy-11 oxoandrographolide to hepatic tissue also decreased after incorporation of 14-deoxy-11 oxoandrographolide into colloidal carriers, as demonstrated by the normal levels of serum alkaline phosphatase (ALP) and serum glutamate pyruvate transaminase (SGPT) in the blood [67]. Interestingly, particle size also proved to be an important factor for drug delivery efficacy. In fact, nanoparticles in a size range below 200 nm have been associated with increased phagocytosis by *Leishmania*-infected macrophages [68].

formulations such as those containing asiaticoside and acaciaside [38], whereas others have a plant origin but were purchased commercially [39]. It should be noted that the most studied species listed in Table 1 are native to developing countries, where the popular use of medici‐

In the last decades, advances in nanoscience have enabled the development of nano-range materials approved for therapeutic use or in clinical development stage. In fact, many encapsulation matrices have been approved by the Food and Drug Administration (FDA) for use in humans, i.e., a wide range of naturally or chemically modified cyclodextrins that are extensively used in medicine and food fields [60, 61]. Similarly, vesicular systems such as liposomes, niosomes, nanoparticles, and microspheres are very useful and have many advantages in delivering drugs of natural origin, representing a promising approach for the treatment of several diseases, including leishmaniasis. Nanoformulations prepared with compounds from medicinal plants and their antileishmanial activity are summarized in Table

**4. Phytocompound nanoformulations and antileishmanial activity**

2. Liposomes, niosomes, and nanoparticles are the main formulations investigated.

Saponins (liposomes) and alkaloids (nanoparticles) are among the main plant constituents used to prepare nanoformulations. Liposomes are mainly formed by a mixture of phosphatidyl choline (PC), cholesterol (Chol), and phosphatidic acid (PA), usually in a 7:4:1 molar ratio [55, 38], whereas nanoparticle formulations have polylactide (PLA) as their main ingredient, providing greater stability, biocompatibility, and an efficient delivery system compared to liposomal structures [50, 57]. The antileishmanial properties of each nanoformulation are

Flavonoids are among the most common phenolic compounds found in the human diet and a variety of members of this family has been described as bioactive agents. Significant antiprotozoal activity of flavonoids has been reported against *Trypanosoma* and *Leishmania* species. Quercetin (Fig. 1) is a widely studied food-derived flavonoid with several biological effects, including antioxidant, antihypertensive, anti-inflammatory, and antiprotozoal activities. Quercetin inhibits parasite arginase activity [62,63] and induces the production of superoxide anion, hydrogen peroxide, and other reactive oxygen species (ROS) by infected cells. Thus, ROS generation induced by quercetin could be crucial for maximal antiparasitic activity, because ROS are naturally generated by macrophages as a mechanism to kill intra‐ cellular parasites such as *Leishmania* [64, 65]. In fact, liposomal, niosomal, microspherulated, and nanocapsulated quercetin formulations have been tested to evaluate the best drug delivery system. In hamster models of *L. donovani* infection, all quercetin vesicular formulations reduced the parasite load compared to the free form of the drug. Nanocapsulated quercetin is more effective than non-capsulated quercetin in the control of leishmaniasis (87% reduction in spleen parasite burden) and its pronounced activity may be related to vesicular composition and size. Moreover, drug efficacy may be inversely correlated to the size of vesicular forms [39].

nal plants is widespread.

356 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

briefly cited below.



LP: liposomes; NS: niosomes; ML: manose-coated liposomes; NP: nanoparticles; MS: microspheres; ME: microemulsions; NG: nanogels.

SC: subcutaneous; IP: intraperitoneal; IV: intravenous.

nr: not reported; nd: not determined.

**Table 2.** Phytocompound nanoformulations and antileishmanial activity.

Triterpenoidic or steroidal saponins directly reduce cell viability via membrane disruption. The mode of action of saponins is related to its aglycone portion, which binds to membrane sterols, leading to the formation of transmembrane pores and loss of intracellular content. This feature of saponins demonstrates the toxic potential of these molecules, hampering their use as antileishmanial agents. Conversely, polymeric nanoparticles composed of PLGA were successfully used to improve the efficacy of β-aescin (Fig. 2), the main saponin isolated from the seeds of horse-chestnut *Aesculus hippocastanumi* (Sapindaceae), lowering its cytotoxic effect for mammalian cells [40]. Bacopasaponin-C (Fig. 2) was firstly reported as an antileishmanial

**Figure 1.** Structure of phenolic compounds loaded in nanoformulations.

**Formulations Active ingredients**

Lipid nanospheres Piperine IV

358 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

Nanoparticles arjunglucoside I SC

Nanoparticles andrographolide *in vitro* assay

Nanoparticles β-aescin *in vitro* assay

Gold nanoparticles quercetin *in vitro* assay

SC: subcutaneous; IP: intraperitoneal; IV: intravenous.

**Table 2.** Phytocompound nanoformulations and antileishmanial activity.

nr: not reported; nd: not determined.

Liposomes asiaticoside nr *L. donovani*

Liposomes acaciaside nr *L. donovani*

bassic acid SC

Niosomes Nanoparticles

Microemulsion Nanoparticles

Nanogels

nanogels.

**Administration Route**

**Target and Parasite reduction**

*L. donovani* (52-90%)

(LP 62%)

(LP 92%)

*L. donovani* (ME 62%; NP 78%)

*L. donovani* (NG 79%; NP75%)

*L. donovani* Axenic amastigotes (IC50 = 36 ± 4 μM/mL) Amastigotes in macrophage (IC50 = 28 ± 2 μM/mL)

*L. infantum*

± 0.23ug/ml)

*L. donovani* Axenic amastigotes (IC50 = 15 ± 3 μM/ml) Amastigotes in macrophage (IC50 10 ± 2 μM/ml)

LP: liposomes; NS: niosomes; ML: manose-coated liposomes; NP: nanoparticles; MS: microspheres; ME: microemulsions; NG:

Triterpenoidic or steroidal saponins directly reduce cell viability via membrane disruption. The mode of action of saponins is related to its aglycone portion, which binds to membrane sterols, leading to the formation of transmembrane pores and loss of intracellular content. This feature of saponins demonstrates the toxic potential of these molecules, hampering their use as antileishmanial agents. Conversely, polymeric nanoparticles composed of PLGA were successfully used to improve the efficacy of β-aescin (Fig. 2), the main saponin isolated from the seeds of horse-chestnut *Aesculus hippocastanumi* (Sapindaceae), lowering its cytotoxic effect for mammalian cells [40]. Bacopasaponin-C (Fig. 2) was firstly reported as an antileishmanial

Amastigotes (IC50 = 1.04

(LP 60%; NI 70%; NP 80%)

**% Entrapment efficiency**

100 [53]

80 [66]

2.8 – 31.9 [40]

77 [69]

60 20

Nd

Nd

100 50

80 60 **References**

[38]

[48]

[57]

agent in 2002, but its mechanism of action remains unclear. This glycoside extracted from *Bacopa monnieri* (Plantaginaceae) has glucose and rhamnose residues attached to the triterpe‐ noid aglycone moiety. The glucose residue may be responsible for targeting bacopasaponin-C to glucose receptors on the cellular surface. Incorporation of bacopasaponin-C into various delivery carriers (niosomes, microspheres, nanoparticles, and liposomes) improved its antileishmanial activity. After six-day treatment with subcutaneous injections of liposomal, niosomal, microencapsulated, or nanocapsulated formulations of bacopasaponin-C, hamster models of *L. donovani* infection showed a significant reduction in spleen parasite burden (81, 86, 79, and 91%, respectively) compared to free drug-treated animals (40%). At the same dose (1.7 mg/kg), the smallest vesicles had the best efficacy, as follows: nanocapsules > niosomes > liposomes > microspheres [44]. Another glycoside with remarkable activity against *Leishma‐ nia* parasites has been isolated from the indigenous plant *Swertia chirata* (Gentianaceae): amarogentin (Fig. 2) is a secoiridoid glycoside with the capacity to inhibit DNA-topoisomerase I, an essential enzyme related to *Leishmania* viability. Liposomal and niosomal formulations of amarogentin (2.5 mg/kg) reduced spleen parasite load by 69 and 90%, respectively, whereas free drug at equivalent dose reduced parasite load by 39%. In addition, both SPTG and ALP activity remained close to normal levels when liposomal or niosomal formulations of this iridoid were used in murine models [55].

The amide alkaloid piperine (Fig. 3) extracted from *Piper nigrum* (Piperaceae), an Indian species commonly used in traditional medicine, has been reported as a potent antileishmanial acting against both visceral (*L. donovani*) and tegumentary (*L. amazonensis*) leishmaniasis [70, 71]. *In vivo* tests have shown that piperine entrapped into liposomes and mannose-coated liposomes were effective against murine models of *L. donovani* infection. After 12-day treatment with liposomal and mannose-coated liposomal formulations of this alkaloid (four doses, 6 mg/mL) administered subcutaneously, a reduction in parasite burden of approximately 77 and 90%, respectively was achieved. Free piperine not only was less effective in reducing parasite burden (29%)but also had higher toxicity to liver compared to the colloidal carriers (ALP = 20.5 µmol of *p*-nitrophenol released/min/dL of sera and SGPT = 77.2 µmol of sodium pyruvate/min/ L of sera) [52]. Nevertheless, better results can be achieved by using lipid nanospheres of piperine (LN-P). A single dose (5 mg/kg) of the lipid formulation composed of stearylamine (LN-P-SA) reduced parasite burden in liver and spleen of *L. donovani-*infected hamsters by 90% and 85% after 15 days post infection, respectively. Despite the size of the vesicles (about 884.6 nm), the efficacy of LN-P-SA may be related to the preferential uptake of stearylamine-bearing

**Figure 2.** Structure of terpenoids loaded in nanoformulations.

positively charged liposomes (+ 24.2 mV) by peritoneal macrophages compared to neutral and negatively charged vesicles [53].

β-carboline alkaloids such as harmane, harmaline, and harmine were initially described as potent psychoactive and hallucinogenic agents. However, a wide range of pharmacological activities have been reported for those compounds, including those against *Leishmania* parasites [72]. Harmine (Fig. 3), isolated from *Peganum harmala* (Nitrariaceae), displays *in vitro* anti-*L. donovani* promastigote activity at 25 µg/mL. Recently, this alkaloid was incorpo‐ rated into liposomes, niosomes, and nanoparticles at an equivalent dose of 1.5 mg/kg body weight, and after six doses administered subcutaneously to *L. donovani*-infected murine models, all harmine-entrapped vesicular formulations were able to reduce spleen parasite burden, especially nanosomes (a reduction of about 79%). Nevertheless, the mechanism of action of harmine against *Leishmania* remains unclear and may be related to necrotic membrane damage.

**Figure 3.** Structure of alkaloids loaded in nanoformulations.

The search for new compounds with antileishmanial activity in medicinal plants is an interesting strategy, however most studies are still limited to basic research and probably only a few compounds will reach stages of clinical trials and development of new drugs. The combination of phytochemistry and nanoformulations may open up new perspectives in the search for new antileishmanial drugs, because drug delivery systems based on nanoformula‐ tions can improve the effectiveness of natural compounds making them more attractive to the development of new therapeutic targets. Due to their structural versatility in terms of size, composition, and ability to incorporate hydrophilic or lipophilic substances, there are many possible applications of nanoparticulate formulations in the treatment and control of leish‐ maniasis.
