Therapeutic Potential of Natural Products as Innovative and New Frontiers for Combating Parasitic Diseases

Abstract

The pressing global challenges of parasitic diseases, particularly prevalent in tropical and subtropical regions, underscore the critical urgent need for innovative therapeutic strategies in identifying and developing new treatments. The immense chemical diversity inherent in nature has rendered natural product (NP) chemistry a promising avenue for the discovery of novel antiparasitic chemotypes. Despite challenges such as sourcing, synthetic complexity, and drug resistance, NPs continue to offer invaluable contributions to antiparasitic therapy. This review focuses on recent advancements in NP chemistry and their application in the development of antiparasitic therapeutics. Key highlights include the identification of new molecular targets such as enzymes, membrane proteins, and metabolic pathways in parasites, as well as the role of metabolomics, genomics, and high-throughput screening in accelerating drug development. Additionally, the exploration of microorganisms (including soil bacteria and fungi) and marine organisms as a latent reserve of bioactive compounds with potent antiparasitic activity is discussed. The review further examines emerging strategies such as chemoinformatics and combination and polypharmacology therapies, aimed at addressing the challenges of antiparasitic chemotherapeutic treatment and advancing the development of new and effective treatments. Ultimately, NP chemistry represents a frontier for the design of novel antiparasitic drugs, offering the potential for more effective and sustainable therapies for combating parasitic diseases.

1. Introduction

Parasitic diseases affect millions of people worldwide, causing significant morbidity and mortality. Despite global efforts, these diseases remain a major public health challenge. Estimates indicate that at any given time, 25% of the world’s population is infected with a parasitic disease, contributing substantially to morbidity, mortality, and disability-adjusted life years (DALYs) [1]. The most vulnerable populations are those in low- and middle-income countries, where inadequate healthcare infrastructure, poor sanitation, and unhygienic conditions are prevalent. Many parasitic diseases result in chronic health conditions, including growth retardation and cognitive impairment in children, as well as organ damage. Beyond health, parasitic diseases impede economic development by reducing productivity, thereby perpetuating a cycle of poverty [1,2].

Major parasitic diseases include malaria, soil-transmitted helminthiases, schistosomiasis, leishmaniasis, and human African trypanosomiasis. Malaria is caused by Plasmodium species and spread through the bites of Anopheles mosquitoes. It continues to be one of the most life-threatening parasitic diseases, recording nearly 263 million malaria cases in the WHO Africa Region in 2023 alone, resulting in an estimated 597,000 deaths. The WHO African Region still bears the heaviest burden, accounting for 94% of malaria cases and 95% of malaria deaths reported in 2023. Tragically, almost 76% of these deaths occurred in children under the age of five [3]. Besides malaria, various parasitic worms, primarily Ascaris lumbricoides (roundworm), Trichuris trichiura (whipworm), Necator americanus and Ancylostoma duodenale (hookworms), are responsible for soil-transmitted helminthiases (STHs). Transmission occurs through contaminated soil, often in areas that lack proper sanitation and hygiene practices. Globally, more than 1.5 billion people are infected with STHs, with the highest prevalence recorded in Asia, sub-Saharan Africa, and South America. Infections can lead to developmental delays in children, anaemia, and malnutrition [4,5,6]. Moreover, schistosomiasis, caused by Schistosoma species (trematode worms), affects over 250 million people worldwide, with nearly 70 million DALYs in Africa, South America, and Asia [7]. The disease is transmitted through contact with freshwater contaminated with larval stages of schistosomes. Chronic infection can cause liver and bladder fibrosis, kidney damage, delayed growth in children, and anaemia [7,8]. Leishmania species, on the other hand, are protozoan parasites transmitted through the bites of infected sandflies, causing leishmaniasis. The disease manifests in three forms: visceral, cutaneous, and mucocutaneous. Visceral leishmaniasis, also known as kala-azar, is the most severe form, characterized by anaemia and weight loss. Skin ulcers resulting from cutaneous leishmaniasis can cause serious disability. The disease prevalence varies by region but is generally associated with insanitary conditions, malnutrition, and population displacement. Annually, nearly 700,000 to 1 million new cases are reported [9]. Human African Trypanosomiasis (HAT), commonly referred to as sleeping sickness, is caused by the protozoan parasite Trypanosoma brucei, transmitted by infected tsetse flies (Glossina). The disease is endemic to sub-Saharan Africa and initially presents with symptoms such as chills, enlarged lymph nodes, joint pains and itching. As the disease progresses, it can cause behavioural changes, poor coordination and disturbances in the sleep cycle. Without treatment, HAT is generally fatal [10,11].

Although less commonly recognized, other parasitic diseases significantly contribute to the overall disease burden. Strongyloidiasis is caused by the parasitic roundworm Strongyloides stercoralis and is transmitted through skin contact with contaminated soil. This disease is endemic in tropical and subtropical regions, especially in Southeast Asia and the Western Pacific [12], and is prevalent in areas with limited access to clean water and sanitation [13]. In immunocompromised individuals, the infection may progress to hyperinfection syndrome, which is potentially life-threatening. Globally, it affects over 600 million people [14]. Cryptosporidiosis, caused by Cryptosporidium species, is a waterborne protozoan disease contributing an estimated 8.37 million DALYs to the global disease burden. It is associated with prolonged episodes of diarrhoea, particularly in children, as well as malnutrition, higher mortality rates, and complications in immunocompromised individuals [15,16]. Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is highly prevalent worldwide, with up to one-third of the global population infected. Foodborne toxoplasmosis alone accounts for approximately 10.3 million cases per year [1,17,18]. Amoebiasis, caused by Entamoeba histolytica, is a protozoan infection common in areas with poor sanitation and can lead to severe intestinal illness [15].

The current range of drugs recommended for the treatment and management of parasitic diseases is limited, and they are predominantly administered through mass drug administration (MDA) programs. Albendazole, 1 and mebendazole, 2 (Figure 1), constitute the primary treatments for STHs. Although 1 is effective against hookworms, it has reduced efficacy against Trichuris trichiura and Ascaris lumbricoides [19,20]. In anticipation of potential drug resistance, the implementation of drug combinations has been recommended, with ivermectin, 3 (A and B), identified as the most promising combination. To further mitigate the growing concern of resistance, new drugs are being developed. Oxantel pamoate, 4 [4], and emodepside, 5 [21], are promising late-stage candidates expected to expand the scope of STH treatments. Praziquantel, 6, remains the primary therapeutic agent for schistosomiasis; however, it only targets adult worms and exerts a limited influence on disease transmission [22]. High reinfection rates in endemic areas underscore the need for new or alternative treatments. Oxamniquine, 7, is employed in areas where 6 (Figure 1) resistance poses a concern [23].

For malaria, artemisinin-based combination therapies (ACTs) are the standard treatment. ACTs comprise an artemisinin derivative—artesunate, artemether or dihydroartemisinin—which acts rapidly to reduce the parasite load, and a partner drug (lumefantrine, mefloquine, amodiaquine, piperaquine), which has a longer half-life and clears remaining parasites to prevent recrudescence. ACTs were designed to delay the development of drug resistance, which was a major issue with earlier monotherapies like chloroquine. Nonetheless, resistance to ACTs is emerging in certain regions, necessitating vigilant monitoring and the exploration of alternative treatments [24]. Tafenoquine, 8, and diminazene aceturate, 9, are also utilized in specific cases, with 8 noted for its effectiveness in preventing malaria relapses [25]. Treatments for leishmaniasis include amphotericin B, 10, and the antimonial compounds miltefosine, 11, and meglumine, 12 [25,26]. The toxicity and resistance associated with antimonial drugs are significant concerns [25]. While 3 is the preferred treatment for strongyloidiasis [27], nitazoxanide, 13, is also the sole proven treatment for Cryptosporidium infections, but it is ineffective in severely immunocompromised patients, and there are limited data on its use in infants [28]. The standard treatment for toxoplasmosis involves a combination of pyrimethamine, 14, and sulfadiazine, 15. Alternatively, trimethoprim, 16, and sulfamethoxazole, 17, can be used [29]. Amoebiasis is typically treated with a combination of metronidazole, 18, or tinidazole, 19, with luminal agents like paromomycin, 20, or diloxanide furoate, 21 (Figure 1) [30].

In all cases, the limited availability of treatment options represents a significant public health challenge. Current treatments, while effective in some instances, face challenges such as drug resistance, limited efficacy, and high reinfection rates. Addressing these issues requires a multifaceted approach, including integrated control strategies, improved healthcare infrastructure, and new drug development. In response to these challenges, the investigation of natural products (NPs) has emerged as a promising avenue for developing new treatments against parasitic diseases [31,32]. NPs have long been recognized for their therapeutic potential, especially in the development of antiparasitic therapies [32,33]. Their diverse chemical structures, unique mechanisms of action, reduced risk of resistance, and accessibility make them valuable candidates for developing new antiparasitic treatments [34]. This review explores the therapeutic potential of NPs as innovative solutions for combating parasitic diseases, emphasizing their historical significance, biological targets, and recent advancements in NP research such as advanced analytical techniques, combination therapy and nanotechnology. It synthesizes the current knowledge on antiparasitic agents derived from plants, microbes, and marine organisms, while examining how the integration of experimental and computational approaches can advance the discovery of more effective and sustainable treatments. The review also addresses key challenges in NP-based drug discovery, including issues related to compound isolation, pharmacokinetics, sustainability, and regulatory hurdles. Furthermore, it outlines future research directions aimed at overcoming these barriers, such as the integration of artificial intelligence and machine learning, the rational design of novel chemical entities inspired by NP scaffolds, strengthening open-access collaborative platforms and the application of polypharmacology. Overall, this review provides a comprehensive framework for leveraging NPs in the development of new antiparasitic therapeutics, with the potential to significantly impact global health outcomes.

2. Methods

A comprehensive literature search was conducted to identify peer-reviewed articles, review papers, and relevant reports on the therapeutic potential of NPs in the treatment of parasitic diseases. The search included publications from January 2000 to July 2025.

The following electronic databases were consulted: PubMed, Scopus, Web of Science and ScienceDirect. Search queries combined both free-text keywords and controlled vocabulary to maximize the retrieval process. Keywords included “natural products”, “plant-derived compounds”, “microbial natural products”, “marine-derived natural products”, “antiparasitic”, “antimalarial”, “antileishmanial”, “antitrypanosomal”, “artemisinin”, “ivermectin”, “combination therapy”, and “drug resistance”. In addition, MeSH terms such as “Antiprotozoal Agents”, “Phytotherapy”, “Drug Resistance, “Parasitic Diseases”, and “Plant Extracts” were used in PubMed to refine the search strategy.

Studies were included if they reported experimental, clinical, or computational findings on NPs with antiparasitic activity; discussed mechanisms of action, pharmacokinetics, or synergistic effects with conventional drugs; and were published in English and in peer-reviewed journals. The exclusion criteria comprised non-peer-reviewed articles, editorials, and commentaries, as well as studies lacking relevance to parasitic diseases or therapeutic applications.

3. Historical Context

Throughout history, various cultures have depended on NPs, primarily derived from herbs, to treat parasitic infections. This dependence emphasizes both the ubiquity of parasites and the need for effective treatments to combat them. Herbal remedies were popular due to their accessibility and perceived safety profiles and were used in various forms such as teas, tinctures or capsules [35]. Meticulous efforts were directed towards the identification of specific plants believed to possess efficacy against parasites. Their application, well documented in ancient texts and traditional medicine practices, has been passed on across generations, offering time-tested inspiration for contemporary healing methods [35].

The evaluation of safety and efficacy regarding these ancient herbal remedies was achieved through close observation of patients administered with medicine. The effects of both successful outcomes and adverse reactions were fastidiously documented, serving as empirical knowledge that has informed current assessments of herbal treatments.

The historical knowledge of the utilization of NPs in traditional medicine systems to treat various parasitic infections provides a valuable foundation for modern scientific research [35,36,37]. NPs can be derived from plants, microorganisms, and marine organisms, providing a vast source of potential antiparasitic agents [38,39,40,41,42,43]. The remarkable structural diversity and unique biological activities exhibited by NPs can be attributed to the natural selection and evolutionary processes that have shaped their utility for millennia. These compounds are usually non-existent in synthetic libraries, enhancing the chances of discovering novel antiparasitic agents.

Notable examples of NPs developed from traditional herbal remedies that have revolutionized the treatment of parasitic infections are quinine, 22, and artemisinin, 23 (Figure 2). A constituent of cinchona tree bark, 22 has been used for centuries to treat malaria. Prior to its discovery, the dried pulverized cinchona bark was mixed with a liquid (typically, alcohol-based) and drunk. The isolation of 22 by Pelletier and Caventou in 1820 marked a significant breakthrough in antimalarial therapy, providing a reliable treatment option long before the advent of modern synthetic drugs [43,44]. Their discovery further marked the first successful application of a chemical compound in the treatment of an infectious disease. Following its isolation, 22 replaced the traditional use of Cinchona bark as the standard treatment for malaria for over a century until its more effective synthetic derivatives, particularly chloroquine, 24 (Figure 2), became available [45]. As resistance to 24 increased, 22 regained importance, especially for treating severe malaria, and it remains an important antimalarial agent in pregnant women and a second-line treatment option [45]. Similarly, the discovery of 23 by Tu Youyou marked another major milestone in addressing resistance to 24 in malaria treatment; 23 was isolated from Artemisia annua, a Chinese traditional medicine long used for managing malaria [46].

Currently, ACTs constitute the first-line treatment option for malaria, culminating in the award of the Nobel Prize in Physiology or Medicine to Tu Youyou [47,48]. Notably, both 22 and 23 benefited significantly from chemical modification of their structures to enhance efficacy or reduce side effects. The introduction of ACTs has significantly reduced malaria incidence and mortality worldwide, showcasing the profound impact of this NP on global health.

The discovery of ivermectin, 3, in the 1970s revolutionized the treatment of parasitic infections in both humans and animals. In pursuit of novel antibiotic-producing microorganisms, Japanese microbiologist Satoshi Ōmura isolated a unique soil bacterium, Streptomyces avermitilis. Through collaboration with William Campbell, a parasitologist at Merck Research Laboratories, promising cultures of S. avermitilis were screened and found to exhibit remarkable antiparasitic activity. The compounds responsible for the observed activity were identified as a class of closely related macrocyclic lactones, subsequently named avermectins. These were chemically modified to produce 3, a more potent and safer derivative. Ivermectin was first introduced for veterinary use in 1981, demonstrating broad-spectrum efficacy against nematodes and ectoparasites in livestock and pets. Following successful clinical trials led by physician Mohammed Aziz in Senegal, which confirmed ivermectin’s effectiveness against onchocerciasis, the drug was approved for human use in 1987 under the name Mectizan^®. Through the Mectizan Donation Program, (Merck & Co., Inc., Rahway, NJ, USA) pledged to provide the drug free of charge to affected populations. The program expanded in 1998 to include lymphatic filariasis and has since delivered billions of treatments, significantly reducing the burden of these two parasitic diseases in endemic regions.

Continued research and development in the field of NP chemistry holds promise for effective and accessible treatments for parasitic infections. The advancement of empirical methods and experimentation has profoundly transformed NP research, evolving it from traditional knowledge to modern scientific inquiry. This transition is marked by the incorporation of sophisticated methodologies for extraction, isolation, and biological screening, high-throughput screening (HTS), and bioactivity-guided fractionation, facilitating efficient identification of bioactive compounds. Employing computational methods has also shifted the focus from identifying NPs based on traditional uses to predicting bioactive compounds with the potential to combat various diseases, including parasitic infections. Cheminformatics and genomics have opened new avenues for discovering secondary metabolites, significantly improving the discovery and validation of natural compounds.

Recent advancements in genomics and metabolomics have identified new molecular targets for antiparasitic drugs. NPs can interact with these targets in unique ways, leading to the development of innovative therapies [49,50,51]. Combining experimental studies with computational techniques, such as virtual screening and molecular docking, enhances the discovery and optimization of natural antiparasitic compounds. This interdisciplinary approach possessed the potential to accelerate antiparasitic drug development [52,53,54].

Additionally, through their novel modes of action, synergistic effects, and the ability to target multiple pathways in parasites, NPs provide alternative pathways to overcome resistance observed in conventional antiparasitic drugs [38,55]. This property is crucial for maintaining the efficacy of treatments and managing resistant parasitic strains, thereby reducing the burden of parasitic diseases and promoting global health security.

4. Biological Targets for Antiparasitic Drug Discovery

The multifunctionality of NPs confers them with the ability to interact with diverse biological targets through various mechanisms, potentially disrupting essential life processes like DNA replication, protein synthesis, reproduction or cellular respiration. These interactions impede reproduction, suppress growth, and eliminate parasites. The identification of plausible biological targets, which are specific molecules within living organisms that drugs bind to produce a desired physiological effect, is a key component in the drug discovery pipeline. A target for drug design against parasitic diseases should be necessary for survival or implicated in disease exacerbation [56]. To overcome off-target effects, targets for antiparasitic drug discovery must not have any orthologues in the human host. These targets are mostly proteins comprising receptors, enzymes, ion channels, and transport proteins or nucleic acids consisting of deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). Advances in omics, especially metabolomics and proteomics, have revealed several metabolic pathways and proteins as biological targets for drug discovery against parasitic infections [57,58]. Some of the notable pathways explored as targets for antiparasitic NPs are outlined.

4.1. Polyamine Biosynthesis

Most disease-causing organisms require the polyamine biosynthesis pathway for their growth and cell differentiation, and hence, it is considered a promising target for drug design against parasitic infections [59]. The pathway involves the catalytic formation of polyamines like putrescine, spermidine, and spermine from basic amino acids. Critical therapeutic targets in the polyamine biosynthetic pathway include ornithine decarboxylase, arginine decarboxylase, S-adenosylmethionine decarboxylase, spermidine synthase, and spermine synthase. Fisetin, 25 (Figure 3), found in fruits like strawberries and grapes, suppressed the growth of both promastigotes and intracellular amastigotes of L. infantum with IC₅₀ values of 0.3 and 0.1 μM, respectively, via arginase inhibition [60]. By employing in silico studies, a di-epoxide derivative, 26, of the natural substance diospyrin, 27 (isolated from Diospyros montana Roxb.), showed non-competitive inhibition of ornithine decarboxylase, exhibiting strong EC₅₀ values of 2.7 µM and 0.18 µM against intracellular amastigotes and promastigotes L. donovani, respectively [61].

4.2. Folate Pathway

Folate biosynthesis, which has attracted attention for antimalarial and other antiparasitic drug discovery, is critical for generating tetrahydrofolate, required for various cellular processes, including nucleic acid synthesis, amino acid metabolism, and methylation reactions [62,63]. Major biological targets present in this pathway include dihydropteroate synthase, dihydrofolate reductase, thymidylate synthase, and 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase, especially for Plasmodium falciparum, the main causative agent for malaria. By inhibiting glutamine amidotransferase/aminodeoxychorismate synthase (folate co-factors), rubreserine, 28 (obtained from the degradation product of physostigmine 29, isolated from Physostigma venenosum), suppressed growth rates of Arabidopsis thaliana, Toxoplasma gondii, and Plasmodium falciparum with IC₅₀ values of 65, 20, and 1 μM, respectively [64]. Virtual screening of 118 sesquiterpene lactones to investigate the dual inhibition of L. major dihydrofolate reductase-thymidylate synthase and pteridine reductase 1 revealed that IC₅₀ values of 6.3 and 4.5 μM, respectively, were required for the identified kaurane, 30, and its derivative, 31 (Figure 3), to attenuate the two targets [65].

4.3. Sterol Metabolism

Ergosterol, a major lipid component of the cell membrane, is required for parasite survival and proliferation. The difference between the parasite’s sterol metabolism and that of the human host renders this pathway an attractive target for selective antiparasitic drug development [66]. Prime targets for antiparasitic drug design include squalene epoxidase, squalene synthase, sterol 14α-demethylase, and sterol C24-methyltransferase. Tomatidine, 32 (Figure 4), isolated from Solanum lycopersicum, exhibited potent and selective antifungal activity against Candida species without causing cytotoxicity to human cells. Employing transcriptional analysis revealed that 32 modulated both C-24 sterol methyltransferase and C-24 sterol reductase, which are key enzymes of the parasite’s ergosterol biosynthetic pathway [67]. In a similar study, six NPs, 6-methoxydihydrochelerythrine, 33, shatavarin IV, 34, plumbagin, 35, 3-α-butyryloxy-boswellic acid, 36, parthenolide, 37, and 12-ursene-3,23-diol, 38, suppressed the growth of L. donovani promastigotes at IC₅₀ values of 0.47, 3.63, 9.59, 13.02, 13.75, and 14.21 μM, respectively. Molecular docking studies corroborated by sterol profiling suggest that the leishmanicidal activities of the NPs were orchestrated via inhibition of C-24 sterol methyltransferase [68]. With an IC₅₀ of 8.14 μM, anydroparthenin 39 (Figure 4), isolated from the leaves of Parthenium hysterophorus, eliminated half the promastigotes of L. donovani by silencing sterol 14α-demethylase and sterol C24-methyltransferase target proteins [69].

4.4. Other Metabolic Pathways

Other targets explored for antiparasitic drug development include purine salvage, trypanothione, and glycolysis pathways. Protozoan parasites are unable to de novo synthesize purine nucleotides and hence salvage purines from the host. The pathway is necessary for maintaining cellular energy and building nucleic acids and hence presents an attractive therapeutic route for the design of antiparasitic chemotypes [70]. The trypanothione pathway, on the other hand, involves the formation of glutathionylspermidine from spermidine and two moles of glutathione molecules, which subsequently lead to trypanothione. Critical for survival and protection against the host’s oxidative stress by providing a unique thiol-redox system, the trypanothione pathway has garnered attention in antiparasitic drug discovery [71]. Glycolysis is a metabolic pathway involving the enzymatic breakdown of glucose to produce pyruvate with the release of energy in the form of adenosine triphosphate (ATP). By providing useful precursors for other metabolic processes, the glycolytic pathway is another viable target for drug discovery against parasitic infections [72]. Attractive biologics explored for antiparasitic drug design targeting these pathways include hypoxanthine-guanine phosphoribosyltransferase, adenine phosphoribosyltransferase, glutathionylspermidine synthetase, trypanothione synthetase, hexokinase, phosphofructokinase, and pyruvate kinase. An in vitro antiparasitic activity evaluation of 81 purine and pyrimidine derivatives identified 40 (IC₅₀ = 19.19 µM) and 41 (IC₅₀ = 18.27 µM) targeting P. falciparum, while 42 (IC₅₀ = 3.78 µM) and 43 (IC₅₀ = 4.24 µM) (Figure 5) were potent against T. cruzi. In silico studies suggested that the antiparasitic target activities observed by these compounds were due to the inhibition of hypoxanthine guanine phosphoribosyltransferase enzymes of P. falciparum and T. cruzi [73]. Ponasik and co-workers identified a bis (trihydro-cinnamoyl) spermidine derivative, kukoamine A, 44, which was first isolated from Lycium chinense, to possess selective Crithidia fasciculata trypanothione reductase inhibition with Ki of 1.8 μM [74]. Employing a combination of in silico and in vitro studies, Zuma et al. observed potent inhibition of iso-mukaadial acetate, 45 (a sesquiterpenoid compound found in the bark of Warburgia salutaris), and betulinic acid, 46 (initially obtained from the bark of Betula pubescens), against selected P. falciparum NF54 strains, recording IC₅₀ values of 1.03 and 1.27 μg/mL, respectively. The antimalarial activity of 45 and 46 (Figure 5) was attributed to the inhibition of P. falciparum glycolytic pathway proteins [75].

Other non-metabolic pathways explored in NP research as antiparasitic agents are also present in this section.

4.5. Cytoskeletal Pathway

The cytoskeleton is a complex network of protein filaments required for movement and structural support and enhances various cellular processes like replication and invasion. It is essential for survival and replication within the host and, therefore, a viable therapeutic option for drug discovery against parasitic infections [76]. Colchicine, 47, vinblastine, 48, and paclitaxel, 49 (Figure 6), are NPs with antiparasitic potential known for interacting with microtubules, which are a major component of the parasite’s cytoskeleton [77,78,79]. By modulating microtubule polymerization or depolymerization, some essential cellular processes like cell division and motility are disrupted.

4.6. Disruption of DNA Replication

For parasitic species, DNA replication is a vital process that is necessary for cell division. Disrupting DNA replication is therefore a common strategy in antiparasitic drug discovery, as that can effectively impede parasite growth and cell death, resulting in successful treatment of parasitic infections. Several enzymes directly involved in DNA replication, serving as therapeutic targets for antiparasitic drug discovery, include DNA polymerase, RNA primase, DNA helicase, DNA ligase, and topoisomerases. Application of high-throughput screening of microbial NP extracts identified PDE-I₂, 50, a non-toxic duocarmycin, 51 (Figure 6) family member with antimalarial activities, recording an IC₅₀ of 18 nM against P. falciparum. Time-of-addition studies indicate that 50 affects parasite DNA metabolism with deformities in DNA replication and chromosome integrity [80]. Kumar and co-workers isolated four NPs from Anthocephalus cadamba, of which only one, cadambine, 52 (IC₅₀ of 10 μM), was found to be potent against DNA topoisomerase IB of Leishmania donovani [81]. A study identified voacamine, 53 (Figure 6), isolated from the plant Tabernaemontana coronaria, to possess leishmanicidal activities, showing a potency of 14.70 μM against a similar target [82].

Despite the identification of NPs with inhibitory activities against these plausible biological targets, there are still promising areas yet to be explored. Notably, disrupting the processes that enable parasites to elude the host’s immune system, employing variable surface glycoprotein or var gene expression, may potentially lead to novel antiparasitic chemotypes. Another area with limited investigation is the food vacuole, which is required for hemoglobin degradation, leading to the release of specific amino acids necessary for parasite growth, offering a therapeutic route for NPs with antiparasitic activities.

5. Key Antiparasitic Natural Products

The search for novel antiparasitic agents continues to accentuate NPs as a prolific source of structurally diverse and biologically active molecules. In recent years, several new compounds exhibiting promising antiparasitic activities have been reported. This section presents the application of experimental methodologies in the identification of NPs from medicinal plants, microorganisms, and marine sources for the treatment of parasitic diseases.

5.1. Plant-Derived Natural Products

Azafluorenone alkaloids. Mass-directed isolation of combined dichloromethane and methanol extracts of dried, ground roots of M. diversifolia yielded two azafluorenone alkaloids: 5,8-dihydroxy-6-methoxyonychine, 54, and 5-hydroxy-6-methoxyonychine, 55 (Figure 7). The alkaloids were evaluated for their antiplasmodial activity against both chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) strains of P. falciparum. Compound 54 exhibited minimal inhibition, with 87% and 80% activity at 120 µM against the 3D7 and Dd2 strains, respectively, and showed no cytotoxicity against HEK293 human embryonic kidney cells at concentrations up to 120 µM. In contrast, 55 showed higher antiplasmodial activity, with IC₅₀ values of 9.9 µM (3D7) and 11.4 µM (Dd2). However, cytotoxicity studies revealed that at 120 µM, 55 inhibited HEK293 cell growth by 96% [83].

Budmunchiamine alkaloids. An investigation of Albizia schimperiana Oliv., a plant native to tropical regions of Africa and traditionally used in the treatment of bacterial and parasitic infections, led to the identification of bioactive antimalarial compounds. Bioassay-guided fractionation of dichloromethane: methanol (1:1) and methanol: water (9:1) extracts from A. schimperiana Oliv. resulted in the isolation of a new macrocyclic spermine alkaloid, along with three known budmunchiamine analogues (56–59) (Figure 7). The isolated compounds displayed promising activity against both the chloroquine-susceptible D6 strain and the chloroquine-resistant W2 strain of P. falciparum, with IC₅₀ values ranging from 120 to 270 ng/mL. Additionally, 56 and 59 exhibited remarkable antileishmanial activity with IC₅₀ values of 1.2 µg/mL and 2.1 µg/mL, respectively, against promastigotes of L. donovani, showing efficacy comparable to the standard pentamidine (2.1 µg/mL), while 58 (0.8 µg/mL) was even more potent than pentamidine [84].

Triterpenoids. The ethyl acetate extract of the root bark of Ziziphus cambodiana, a plant widely distributed in northeast Thailand and traditionally used to treat infections, was investigated for its antiplasmodial activity. Bioassay-guided isolation yielded several triterpenoid compounds active against P. falciparum, including 3-O-vanillylceanothic acid, 60 (IC₅₀ = 3.7 µg/mL), betulinaldehyde, 61 (IC₅₀ = 6.5 µg/mL), 2-O-E-p-coumaroyl alphitolic acid, 62 (IC₅₀ = 0.9 µg/mL), and zizyberenolic acid, 63 (IC₅₀ = 3.0 µg/mL). Structure–activity relationship analysis indicated that vanillyl and coumarate moieties enhanced activity. Furthermore, the presence of a double bond in ring A, as seen in zizyberenalic acid, 64, improved potency compared to its saturated analogue, zizyberanalic acid 63 (Figure 7), which was inactive in the antiplasmodial assays [85].

Conessine. Holarrhena antidysenterica has been traditionally used in Chinese medicine and Ayurveda for the treatment of malaria, amoebiasis, and various other diseases [86]. In vitro evaluation of conessine, 65, a steroidal alkaloid isolated from H. antidysenterica, revealed notable antiplasmodial activity. The compound exhibited IC₅₀ values of 1.9 µg/mL in the schizont maturation assay and 1.3 µg/mL in the parasite lactate dehydrogenase (pLDH) assay. However, 65 also displayed cytotoxicity toward host cells, with an IC₅₀ value of 14 µg/mL [87].

Rhaphidophora decursiva. Bioassay-guided fractionation of the chloroform extract from the leaves and stems of R. decursiva led to the isolation of several bioactive antimalarial compounds. Among these, polysyphorin, 66 exhibited IC₅₀ values of 404 ng/mL and 368 ng/mL against the D6 (chloroquine-sensitive) and W2 (chloroquine-resistant) strains of P. falciparum, respectively. Additionally, rhaphidecurperoxin, 67, demonstrated IC₅₀ values of 540 ng/mL (D6 strain) and 420 ng/mL (W2 strain). Both compounds also showed promising in vivo antimalarial efficacy, with ED₅₀ values of 2 µg/mL for 66 and 4 µg/mL for 67 (Figure 7) [88].

Cyclopeptide alkaloids. Compounds 68 and 69 (Figure 8), isolated from the dichloromethane extract of the stem bark of Ziziphus mauritiana, exhibited antiplasmodial activity with IC₅₀ values of 15.1, 14.7, and 2.6 µM, respectively. Structure–activity relationship analysis revealed that the presence of a methoxy group at the C-14 position in these cyclopeptides enhanced antiplasmodial potency. In addition, the introduction of an indole substituent at C-27 in compounds 70 and 71 was crucial for activity against T. b. rhodesiense, displaying IC₅₀ values of 12.7 and 6.4 µM, respectively [89].

Tryptophan esters. The dichloromethane and methanol extracts of Bidens pilosa were investigated for their antitrypanosomal activity against bloodstream forms of Trypanosoma brucei, which displayed IC₅₀ values of 3.29 and 5.86 µg/mL, respectively. Bioactivity-guided fractionation and chromatographic separations led to the isolation of two tryptophan esters, 72 and 73 (Figure 8), as the active constituents, which exhibited IC₅₀ values of 0.66 and 1.46 µg/mL, respectively, while maintaining relatively low cytotoxicity [90]. Further studies revealed that both compounds induced apoptosis-like cell death in trypanosomes, underscoring their potential as antitrypanosomal agents [91].

Vitellaria paradoxa. The ethyl acetate and water extracts of the powdered stem bark and seeds of V. paradoxa were evaluated for their trypanocidal effects against T. brucei brucei. Bioassay-guided isolation from the ethyl acetate extract led to the identification of cyclitol, 74 (IC₅₀ = 56.98 µM), pentadecanoic acid, 75 (IC₅₀ = 70.13 µM), and 1α,2β,3β,19α-tetrahydroxyurs-12-en-28-oic acid, 76 (IC₅₀ = 11.30 µM) (Figure 8), as active constituents. Given that Human African Trypanosomiasis (HAT) is associated with excessive production of proinflammatory cytokines, the antioxidant properties of V. paradoxa may provide a pharmacological rationale for its traditional use in HAT management [92].

Pothomorphe umbellata. Extracts from the roots of P. umbellata, prepared using ethanol:water (80:20, v/v) and n-hexane, demonstrated schistosomicidal activity against adult Schistosoma mansoni, with complete worm mortality observed with EC₅₀ values of 18.7 and 9.2 µg/mL, respectively. Bioassay-guided fractionation of the active n-hexane extract led to the isolation of 4-nerolidylcatechol (4-NC), 77 and peltatol A–C, 78–80 (Figure 8). Among these, 77 exhibited the highest potency, with an EC₅₀ of 0.91 µg/mL and no detectable cytotoxicity to mammalian cells. In vivo studies showed that oral administration of 4-NC reduced adult worm burden by 52.1–52.3% in infected mice. The moderate efficacy observed may be attributed to its low oral bioavailability. Notably, unlike praziquantel, 6 (Figure 1), 77 also exhibited significant activity against juvenile S. mansoni, reducing the worm burden by 52.4%. Structural modifications to improve bioavailability may further enhance its therapeutic potential [93].

Lignan derivatives. Two lignan derivatives, dehydrodieugenol B, 81 and methyl dehydrodieugenol B, 82 (Figure 8), were isolated from the Brazilian plant Nectandra leucantha and evaluated for their antischistosomal activity. 81 demonstrated notable activity against adult Schistosoma mansoni, with an EC₅₀ value of 31.9 µM, while exhibiting low cytotoxicity towards mammalian cells, indicating a favourable safety profile. In contrast, its methylated analogue, 82, was inactive, emphasizing the critical role of a free phenolic hydroxyl group at the 4-position for biological activity. Although the precise mechanism of action in schistosomes remains to be elucidated, studies in trypanosomatids suggest that 81 may exert its effects through transient depolarization of the plasma membrane potential and disruption of intracellular calcium homeostasis [94].

Licochalcone A analogues. Licochalcone A, 83 (Figure 9), a natural chalcone isolated from the roots of Chinese liquorice plants (Glycyrrhiza glabra and Glycyrrhiza inflata), has garnered considerable interest for its potent antileishmanial properties. Mechanistic studies have shown that 83 primarily targets mitochondrial function in Leishmania species by inhibiting mitochondrial dehydrogenases and disrupting the respiratory chain, resulting in impaired energy metabolism and parasite death. These effects have been demonstrated against the promastigote forms of Leishmania major and Leishmania donovani, underscoring its potential as a lead compound for antileishmanial therapy [95]. Further investigations evaluated 83 and its synthetic analogues, 84 and 85, against Leishmania amazonensis and Leishmania infantum. 83 exhibited potent leishmanicidal activity against L. amazonensis promastigotes, with IC₅₀ values of 20.26 µM and 3.88 µM after 24 and 48 h of treatment, respectively, and against amastigote forms with an IC₅₀ of 36.84 µM after 48 h. In contrast, analogue 84 showed modest activity, with IC₅₀ values of 79.94 µM and 67.16 µM at 24 and 48 h, respectively, while 85 (Figure 9) was largely inactive (IC₅₀ > 300 µM). Against L. infantum, 83 displayed IC₅₀ values of 41.10 µM and 12.47 µM at 24 and 48 h, respectively, for promastigotes and an IC₅₀ of 29.58 µM against amastigote forms after 48 h. Cytotoxicity assays on mammalian cells revealed a CC₅₀ of 132.21 µM, indicating a reasonable therapeutic window. In vivo efficacy was further investigated following oral administration of 3.4.1 at doses of 20 and 50 mg/kg, resulting in a significant reduction in parasite burden in the liver by 15.27% and 43.67%, respectively, and in the spleen by 20.69% and 39.81%, respectively [96].

Camellia sinensis. C. sinensis, the source of green tea, is widely consumed for its diverse health benefits. The petroleum ether fraction of C. sinensis, containing (−)-gallocatechin, 86, (−)-epigallocatechin, 87, (−)-gallocatechin gallate, 88, and (−)-epigallocatechin gallate, 89 (Figure 9), has shown inhibitory effects against the promastigote forms of Leishmania amazonensis and Leishmania braziliensis. Among these, 89 exhibited the most potent antileishmanial activity, primarily through the generation of reactive oxygen species (ROS), leading to mitochondrial dysfunction and parasite death [97]. In vivo, a hydrophilic ointment containing 15% 89 significantly reduced lesion size and parasite burden in a cutaneous leishmaniasis model, achieving an inhibition rate of 80.4% after 18 days of daily topical application compared to untreated controls [98]. Additionally, 89 inhibits arginase, a key enzyme in the polyamine biosynthesis pathway critical for parasite growth and survival, further contributing to its antileishmanial efficacy [99].

Thiophene derivatives. Crude dichloromethane extract, its fractions, and compounds isolated from the aerial parts of Porophyllum ruderale, a plant native to Brazil, were evaluated for their antileishmanial activity. The crude extract was found to be active against both the promastigote and axenic amastigote forms of Leishmania amazonensis, with IC₅₀ values of 60.3 µg/mL and 77.7 µg/mL, respectively. Further bioassay-guided fractionation led to the isolation of two thiophene derivatives: 5-methyl-2,2′:2″-terthiophene, 90 and 5′-methyl-[5-(4-acetoxy-1-butynyl)]-2,2′-bithiophene, 91. These compounds exhibited significantly enhanced activity, with IC₅₀ values of 7.7 µg/mL and 19.0 µg/mL for 90, and 21.3 µg/mL and 28.7 µg/mL for 91 (Figure 9), against the promastigote and axenic amastigote forms, respectively [100]. Mechanistic studies revealed that treatment with these thiophene derivatives resulted in compromised mitochondrial membrane integrity, as assessed by flow cytometry and confirmed by transmission emission spectroscopy. Furthermore, scanning electron microscopy showed morphological changes, including cell rounding and swelling, indicating substantial structural damage to the parasites [101].

Khaya senegalensis. The stem bark of Khaya senegalensis, a medicinal plant native to Cameroon, was investigated for its antileishmanial activity against promastigote forms of Leishmania donovani. Both the crude extract and its fractions exhibited moderate to potent activity, with IC₅₀ values ranging from 5.99 µg/mL to 2.68 µg/mL. Isolated compounds displayed a range of activities, with IC₅₀ values between 81.73 µg/mL and 6.43 µg/mL. Notably, Bellericagen B, 92 (6.50 µg/mL), gynuramide IV, 93 (6.43 µg/mL), and a combination of β-sitosterol glycoside, 94 and β-stigmasterol glycoside, 95 (9.60 µg/mL) were identified as key contributors to the observed antileishmanial effects [102].

Cephaeline. The ethanolic extract of Psychotria klugii, a plant widely distributed in tropical America and the West Indies, yielded cephaeline, 96, an isoquinoline alkaloid that exhibited significant antileishmanial activity against Leishmania donovani promastigotes. The IC₅₀ of 96 was found to be 0.03 µg/mL, making it 20-fold more potent than pentamidine, 97 (IC₅₀ 0.7 µg/mL) and 5-fold more potent than amphotericin B 10 (IC₅₀ 0.17 µg/mL), which are both first-line treatments for leishmaniasis [103].

Xanthone and naringeninyl biflavonoid. 1,4,5-Trihydroxy-3-(3-methylbut-2-enyl)-9H-xanthen-9-one, 98 (Figure 10), isolated from the root bark of Garcinia livingstonei, when tested against P. falciparum, L. infantum and T. brucei, demonstrated an IC₅₀ of 27.10 and 0.87 µM, respectively. In addition, the biflavonoid ent-naringeninyl-(I-3α, II-8)-4′-O-methylnaringenin, 99, displayed notable activity against P. falciparum with an IC₅₀ value of 6.0 µM [104].

Conyza filaginoides. C. filaginoides has been traditionally used in Mexico for the treatment of gastrointestinal diseases. Bioassay-guided fractionation of its antiprotozoal extract led to the isolation of several bioactive compounds with promising activity. Among these, erythrodiol, 100 and nicotiflorin, 101, were found to be active against both Entamoeba histolytica and Giardia lamblia, exhibiting IC₅₀ values of 12.7 and 30.9 µg/mL, respectively. Additionally, quercetin 3-O-(6″-O-E-caffeoyl)-β-D-glucopyranoside, 102 and astragalin, 103 showed significant antiamoebic activity against the trophozoites of E. histolytica, with IC₅₀ values of 12.0 and 14.7 µg/mL, respectively. Furthermore, isorhamnetin 3-O-(6″-O-E-caffeoyl)-β-D-galactopyranoside, 104 (Figure 10), demonstrated notable activity against G. lamblia, with an IC₅₀ value of 15.3 µg/mL [105].

Juliprosopine. Branco et al. evaluated the in vitro anthelmintic activity of the alkaloidal fraction from Prosopis juliflora pods against gastrointestinal nematodes in goats. The alkaloidal fraction obtained from the ethyl acetate extract demonstrated strong ovicidal activity, with IC₅₀ and IC₉₀ values of 1.1 mg/mL and 1.43 mg/mL, respectively. However, the fraction exhibited low activity and high toxicity at the larval stage. Juliprosopine, 105, a piperidine alkaloid, was identified as the major constituent of the alkaloidal fraction, and findings suggest that it may be particularly valuable for early-phase treatment of gastrointestinal nematode infections [106].

Neolignans. Neolignans threo-austrobailignan-6, 106 and verrucosin, 107, isolated through bioassay-guided fractionation of methanolic extracts of Saururus cernuus, were tested against adult worms of S. mansoni. Both compounds induced significant tegumental damage in adult worms, with EC₅₀ values ranging from 12.6 µM to 28.1 µM. In addition to disrupting the worm surface structure, they also reduced egg production and exhibited low cytotoxicity toward mammalian cells [107].

Perylenequinones. Scutiaquinone A, 108 and scutiaquinone B, 109 (Figure 10), aromatic polycyclic perylenequinones isolated from the roots of Scutia myrtina [108], were evaluated as potential inhibitors of Schistosoma mansoni glutathione S-transferase (GST), an essential detoxification enzyme. GST plays a crucial role in parasite survival by mediating the neutralization and elimination of harmful metabolites. It was hypothesized that inhibition of GST would compromise the parasite’s detoxification capacity, leading to toxic metabolite accumulation, disruption of redox homeostasis, and eventual parasite death. Both compounds exhibited strong binding affinity for GST, underscoring the potential of NPs to disrupt critical biochemical pathways in parasitic organisms [109].

Calliandra portoricensis, Siphonochilus aethiopicus and Abrus precatorius. An investigation of the antitrypanosomal activity of Nigerian plants led to the isolation of compounds from the roots of C. portoricensis, bulbs of S. aethiopicus, and seeds of A. precatorius. Extracts of S. aethiopicus yielded isofuranodienone, 110, chloranthene F, 111, curzerenone, 112, epi-curzerenone, 113, and 8(17)-12E-labdadiene-15,16-dial, 114, with EC₅₀ values ranging from 4.31 µg/mL to 7.09 µg/mL. Extracts of C. portoricensis yielded spinasterone, 115 (EC₅₀ = 6.35 µg/mL), lupeol, 116 (EC₅₀ = 13.37 µg/mL), and linalolic acid, 117 (EC₅₀ = 3.35 µg/mL). From A. precatorius, abruquinone B, 118 (Figure 10), was isolated with an EC₅₀ value of 16.70 µg/mL [110].

Acroptilon repens. In efforts to control the vectors responsible for malaria transmission, several countries have adopted larval control strategies targeting Anopheles mosquitoes. In this context, plant-derived compounds have emerged as eco-friendly alternatives to synthetic insecticides. Notably, essential oils extracted from Acroptilon repens have demonstrated significant larvicidal activity against Anopheles stephensi [111]. These oils, obtained via hydrodistillation using a Clevenger apparatus, were analyzed using GC-MS, which identified major constituents such as caryophyllene oxide, 119, α-cubebene, 120, 1-heptadecene, 121, δ-cadinene, 122, and β-cubebene, 123 (Figure 11). Larvicidal bioassays revealed LC₅₀ and LC₉₀ values of 7 ppm and 34 ppm, respectively, for the A. repens essential oils [112].

Hyptis sauveolens Kuntze and Momordica charantia. Extracts from the leaves of Hyptis sauveolens Kuntze and Momordica charantia were tested against trypomastigotes of T. brucei brucei and Trypanosoma congolense. The most promising EC₅₀ values were obtained for the purified sauveolol 124 fraction, which exhibited an EC₅₀ of 2.71 µg/mL, and sauveolic acid 125, which showed an EC₅₀ of 1.56 µg/mL against T. brucei brucei [113].

Machaeridiol B. The ethanolic extract of Michaerium multiflorum yielded machaeridiol B, 126, which exhibited significant antileishmanial activity against Leishmania donovani with IC₅₀ and IC₉₀ values of 0.9 µg/mL and 8.0 µg/mL, respectively, demonstrating the potential of M. multiflorum as a source of bioactive compounds for leishmaniasis treatment [114].

Toosendanin. Toosendanin, 127, a triterpenoid compound isolated from Melia toosendan Sieb. et Zucc. and Melia azedarach L., has been identified as the principal bioactive constituent responsible for their traditional anthelmintic properties. These plants have long been used in Chinese medicine, particularly against Ascaris infections. Clinical studies have shown that oral administration of toosendanin results in a high Ascaris expulsion rate of 91.23%, with minimal adverse effects reported [115].

Tetracyclic iridoids. Three tetracyclic iridoid compounds, 128, molucidin, 129, and 130 (Figure 11), were isolated from the chloroform fraction of Morinda lucida leaves, and they exhibited potent activity against the GUTat 3.1 strain of Trypanosoma brucei brucei, with IC₅₀ values of 1.27, 3.75, and 0.43 µM, respectively. Compounds 128 and 130 suppressed the expression of paraflagellar rod and protein subunit 2 (PFR 2), leading to cell cycle disruption and induction of apoptosis in bloodstream forms of the parasite [116].

Piperine, Curcumin, quercetin. The leishmanicidal potential of the NPs piperine, 131, curcumin, 132, and quercetin, 133, all widely recognized for their broad antiparasitic activities, was evaluated. An in vitro study using macrophages infected with Leishmania braziliensis amastigotes showed that 131 exhibited remarkable activity, with an EC₅₀ value of 8.8 µM, significantly reducing intracellular parasite viability. Compounds 132 and 133 also demonstrated leishmanicidal effects, with EC₅₀ values of 30.9 µM and 32.0 µM, respectively. All three compounds displayed low cytotoxicity. Furthermore, topical application of castor oil formulations containing these compounds in golden hamsters resulted in a 100% therapeutic response for 133 quercetin-treated lesions, while those containing 132 and 131 achieved therapeutic responses of 83% and 67%, respectively [117]. When 132 was evaluated for its antischistosomal activity against adult worms of Schistosoma mansoni and S. haematobium, a time- and concentration-dependent effect was observed. At a concentration of 500 µM, 132 achieved 100% mortality of adult worms of both S. mansoni and S. haematobium within 2 h of incubation. At a concentration of 250 µM, all S. haematobium worms were killed within 2 h, while 88% of S. mansoni worms died within 4 h. Furthermore, increasing concentrations of 132 (Figure 11) (starting from 50 µM) led to varying degrees of tegumental damage, ranging from surface swelling in different parts of the integument to pronounced edema, collapsed tubercles, and extensive tegumental deformation with loss of other intertubercular structures [117].

Lupanine and Sparteine derivatives. Given the growing concern over drug resistance in nematodes and its detrimental impact on ruminant farming, the Charvet and Sallé group explored the potential of nutraceuticals—dietary supplements—as alternatives to control parasitic infections. The group specifically investigated seed extracts from Lupinus species and found that they exhibited significant anthelmintic activity against major ruminant trichostrongylids Haemonchus contortus and Teladorsagia circumcincta. Further investigation revealed an antagonist mode of action, with electrophysiological data indicating the alkaloids inhibit nematode acetylcholine receptors through both competitive and non-competitive mechanisms [118].

Octyl gallate and estradiol benzoate. Several promising bioactive compounds have shown significant activity against Toxoplasma gondii. Among them, octyl gallate, 134, and estradiol benzoate, 135 (Figure 11), both isolated from marine sources, inhibited tachyzoite growth by over 70%, with IC₅₀ values of 4.41 µM and 5.66 µM, respectively. Flow cytometric analysis suggested that their anti-T. gondii activity might stem from the inhibition of tachyzoite progression from the G to the S phase of the cell cycle [119].

5.2. Microbial Natural Products

Avermectins. Avermectins, 3, were first discovered by Satoshi Ōmura in 1973 through the fermentation of the actinomycete Streptomyces avermectinius. These compounds were identified as 16-membered macrocyclic lactones belonging to the class of endectocides, due to their ability to kill a broad range of disease-causing organisms and vectors of pathogens. A semi-synthetic derivative with enhanced efficacy was subsequently developed and consisted of a mixture of approximately 80% 22,23-dihydroavermectin B_1a, 136, and 20% 22,23-dihydroavermectin B_1b, 137 (Figure 12). 3 exhibited broad-spectrum activity against various insects, acarines, and nematodes and proved highly effective against intestinal worms, Onchocerca volvulus, and lymphatic filariasis. Partial elucidation of its mode of action revealed that 3 primarily acted as an agonist of glutamate-gated chloride channels, disrupting the nervous system and muscle function of parasites. Furthermore, at low concentrations, 3 was shown to interfere with the reproductive mechanisms of female worms [120].

Prodiginines. Prodiginines, a family of red-pigmented linear and cyclic oligopyrroles produced by actinomycetes and other eubacteria, have emerged as promising antimalarial candidates. In vitro assays against the Plasmodium falciparum D6 strain demonstrated potent activity, with prodigiosin, 138, undecylprodiginine, 139, metacycloprodiginine, 140, and streptorubin B, 141 (Figure 12) exhibiting IC₅₀ values of 8, 7.7, 1.7, and 7.8 nM, respectively, outperforming the reference drug chloroquine, 24 (IC₅₀ = 11 nM). Although the precise mode of action remains unclear, structure–activity relationship (SAR) studies of synthetic analogues revealed that non-alkylated terminal pyrroles are essential for maintaining antimalarial potency. Furthermore, strategic incorporation of alkyl or aryl substituents at positions 3 and 5 of the pyrrole core significantly enhanced activity, yielding IC₅₀ values of ≤1 nM. Notably, analogues bearing aryl groups on the pyrrole ring displayed the most favourable profiles, achieving a 92% parasite reduction at a dosage of 5 mg/kg/day and a 100% reduction at 25 mg/kg/day in Plasmodium yoelii murine infection models, without any apparent weight loss or observable signs of toxicity [121].

Macrolide glycoside alkaloids. The marine cyanobacterium Okeania sp., collected from Akuna Beach, Okinawa, Japan, was investigated for its antitrypanosomal potential. From the ethanolic extract, four macrolide glycoside alkaloids, akunalides A–D, 142–145 (Figure 12), were isolated and exhibited moderate growth inhibitory activity against the bloodstream forms of Trypanosoma brucei rhodesiense, with IC₅₀ values ranging from 11 µM to 14 µM, indicating their potential as lead structures for antitrypanosomal drug development. Notably, akunalide A, 142 and akunalide C, 144 displayed no cytotoxicity at concentrations up to 150 µM [122]. In a separate study, polycarvernoside E, 146 was also isolated from the ethanolic extracts of Okeania sp. and was found to exhibit moderate activity against the bloodstream form of T. b. rhodesiense, with an IC₅₀ value of 9.9 µM [123]. Further investigations by Suenaga et al. revealed a long-chain fatty acid linear lipopeptide, ouhanamide, 147, which showed potent activity, with IC₅₀ values of 1.2 µM against T. b. rhodesiense and 4.2 µM against Plasmodium falciparum, and did not exhibit cytotoxicity to HeLa cells at 10 µM [123].

Paenidigyamycin A. Paenidigyamycin A, 148, isolated from Paenibacillus sp. strain DE2SH obtained from mangrove rhizosphere soils in Ghana, has demonstrated significant antiparasitic activity. In Schistosoma mansoni cercariae, 148 induced complete mortality within 1 h at concentrations of 25–100 µM. In addition to its antischistosomal effects, 148 exhibited notable antileishmanial activity. When tested against Leishmania major promastigotes, it showed an IC₅₀ value of 0.75 µM, 3-fold less potent compared to the reference drug amphotericin B, 10 (Figure 1) (IC₅₀ = 0.31 µM). However, its activity against Leishmania donovani was lower, with an IC₅₀ of 7.02 µM. The presence of an imidazole moiety in 148 has been proposed to be critical to its antiparasitic effects, facilitating interactions with various enzymes and receptors and contributing to its broad-spectrum efficacy [124].

Leucinostatins. A high-throughput screening of a library of NPs from soil fungi sources and subsequent bioactivity-guided fractionation led to the identification of leucinostatins, 149–153 (Figure 12), isolated from the soil fungus Ophiocordyceps sp. They demonstrated promising activity against the intracellular amastigote form of Trypanosoma cruzi, with EC₅₀ values ranging from 2.8 to 12.0 nM and no detectable toxicity against host cells. These compounds were confirmed to inhibit kinetoplastid T. cruzi, and recent structure–activity relationship (SAR) studies involving leucostatin A, 149 against T. brucei and Leishmania donovani further supported their antiparasitic potential [125]. In a separate study investigating activity against T. brucei, it was reported that 149 and leucostatin B, 150 (Figure 12), target ATP synthase and/or disrupt Ca²⁺ and pH homeostasis. The antitrypanosomal activity in T. cruzi has been attributed to the destabilization of the parasite’s inner mitochondrial membrane [126].

5.3. Marine-Derived Natural Products

Aplysinella rhax. A modified Kupchan partitioning method was applied to the marine sponge extract Aplysinella rhax, followed by further fractionation using reversed-phase solid-phase extraction and reversed-phase HPLC. This process yielded psammaplin A, 154 (Figure 13), which exhibited IC₅₀ values of 30 µM and 60 µM against Trypanosoma cruzi and Plasmodium falciparum, respectively. Its biphenylic dimer, bisaprasin, 155, showed moderate activity, with corresponding IC₅₀ values of 19 µM and 26 µM [127].

Isoliolide. Bioassay-guided fractionation of Macrorhynchia philippina led to the isolation of isoliolide, 156, from the methanolic extract of the cnidarian, which was investigated for its antitrypanosomal activity against the trypomastigote and amastigote forms of Trypanosoma cruzi. 156 exhibited IC₅₀ values of 31.9 and 40.4 µM, respectively, and demonstrated no cytotoxicity up to 600 µM. The authors hypothesized that 156 induces irreversible damage through the loss of the mitochondrial membrane or an apoptosis-like mechanism, accompanied by disruption of the plasma membrane, suggesting a mechanism of action similar to 10 (Figure 10) in Leishmania. This membrane disruption was proposed to result in early mitochondrial dysfunction or necrosis [128].

Sulfoquinovosyldiacylglycerol. Investigation of the antiprotozoal activity of whole extracts from the brown alga Lobophora variegata revealed activity against Trichomonas vaginalis (IC₅₀ = 3.2 µg/mL), Giardia intestinalis (10.5 µg/mL), and Entamoeba histolytica (10.8 µg/mL). Isolation of compounds from the active non-polar fractions yielded 1-O-palmitoyl-2-O-myristoyl-3-O-(6‴-sulfo-α-D-quinovopyranosyl)-glycerol, 157, together with small amounts of 1,2-di-O-palmitoyl-3-O-(6‴-sulfo-α-D-quinovopyranosyl)-glycerol, 158, and 1-O-palmitoyl-2-O-oleoyl-3-O-(6‴-sulfo-α-D-quinovopyranosyl)-glycerol. A combination of compounds 157–158 displayed synergistic activity with an IC₅₀ value of 3.9 µg/mL against E. histolytica and moderate activity against T. vaginalis (8.0 µg/mL) [129].

Plakortide P. Plakortide P, 159, isolated from the methanolic extract of the marine sponge Plakortis angulospiculatus, exhibited potent activity against Leishmania chagasi, with IC₅₀ values of 1.9 µg/mL against promastigotes and 0.5 µg/mL against intracellular amastigotes, with an excellent safety profile. Furthermore, 159 (Figure 13) demonstrated significant activity against the trypomastigote stage of Trypanosoma cruzi, with an IC₅₀ value of 2.3 µg/mL, making it approximately 15-fold more potent than the standard drug benznidazole [130].

6. Advances in Natural Product Research as Potential Antiparasitic Agents

6.1. Advances Made in Isolation, Purification, Elucidation and Biological Assay of Natural Products

In recent decades, substantial progress has been achieved in NP research. The rapid pace of technological advancements and the growing availability of data have paved the way for innovative methods to enhance the efficiency of NP isolation, purification, elucidation, and biological assays.

Hyphenated techniques, which integrate chromatographic purification methods with structural elucidation techniques, have emerged as essential tools for addressing complex analytical challenges. Techniques like Liquid Chromatography–Mass Spectrometry (LC-MS) and Liquid Chromatography–Nuclear Magnetic Resonance (LC-NMR) facilitate online structure elucidation without the need for prior isolation, thereby speeding up the identification of bioactive compounds. LC-MS combines the separation power of liquid chromatography with the detection capabilities of mass spectrometry, while LC-NMR offers detailed structural insights through nuclear magnetic resonance [131]. DOSY NMR paired with DEREP-NP databases has also enhanced the efficiency of dereplicating known compounds and swiftly identifying novel NPs within complex mixtures [132].

The isolation process is a crucial step in NP drug discovery; however, the presence of small quantities and the rediscovery of known NPs pose new challenges in targeting and dereplication. Isolation methods have evolved remarkably, with current approaches employing powerful metabolite profiling before isolation work [133]. For example, analytical platforms that combine ultra-high-performance liquid chromatography (UHPLC) for high-resolution, high-throughput chromatographic separations with a high-resolution mass spectrometer capable of data-driven acquisition to produce high-resolution mass spectrometry (HRMS) and tandem mass spectrometry (MS/MS) spectra have been employed for detailed compound annotation. Such an approach allows for putative structural annotation of most detected metabolites for automated early identification of known NPs (dereplication) and enables rapid estimation of extract composition before the targeted isolation of prioritized NPs [134]. The effective integration of these novel approaches into workflows for isolating NPs significantly streamlines the process of obtaining pure NPs from complex biological matrices, often in a single step and through suitable extract enrichment.

Biological assays play a vital role in deciphering the mechanisms of action and assessing the therapeutic potential of NPs. The conventional bioassay-guided isolation method involves repeated testing of subfractions at various levels, a process that is often time-consuming, labour-intensive, and expensive. This approach frequently results in the re-isolation of known compounds, leading to disappointing outcomes. In recent years, the advent of omics approaches has offered a comprehensive perspective on the biological effects of NPs, providing detailed insights into their mechanisms of action and potential therapeutic applications [131]. Advances in omics analyses, such as genomics, transcriptomics, proteomics, and metabolomics, generate complex multivariate datasets that require computational and chemometric tools for interpretation. Genomic studies have revealed that microorganisms possess a much greater potential to produce novel and complex secondary metabolites [135]. Genome-mining techniques have been developed to activate silent biosynthetic gene clusters in microorganisms, enabling the discovery of cryptic NPs that remain undetected under conventional laboratory conditions [132].

The application of computational platforms, including bioinformatics and multivariate statistical tools, facilitates the use of omics multidata to elucidate pathophysiological effects, target specificity, and molecular impacts, as well as characterize the pharmacodynamics, pharmacokinetics, and toxicology of NPs and their compounds [136]. During the drug discovery process, applications such as docking and virtual screening can leverage novel machine learning algorithms, including deep learning. Machine learning methods enable the virtual screening of thousands of compounds, utilizing data from high-throughput screening [137]. Tools like SMART, DeepSAT, and HMBC networks have been developed to automate the annotation of NP structures by harnessing information from 2D NMR experiments and machine learning algorithms. Strategies that integrate bioactivity data with NMR and MS analysis, such as ELINA, Plasmodesma, and MADBYTE, can help identify minor bioactive compounds that might be overlooked in traditional bioactivity-guided fractionation approaches, permitting the study of scaffolds present at trace levels and potentially leading to the discovery of new drug leads from natural sources [131].

These advancements, including computational studies, have significantly expanded the scope of NP research, offering new opportunities for discoveries in the field.

6.2. Computational Studies

In recent years, in silico studies have become an essential strategy in NP research, revolutionizing the methodologies through which scientists discover, analyze, and develop novel compounds with antiparasitic potential. These computational approaches enable rapid exploration of vast chemical libraries, prediction of bioactivity, and rational design of lead compounds, significantly accelerating the drug discovery pipeline [138]. By contrast, traditional screening methods, which rely on empirical biological assays, offer distinct advantages in identifying therapeutic candidates with activity against whole parasites [139,140]. These methods do not require prior knowledge of a compound’s mechanism of action, allowing for the discovery of broad-spectrum agents capable of engaging in complex biological interactions. Furthermore, traditional screening can yield diverse drug-like chemotypes with proven ability to reach and interact with parasitic targets in vivo [140]. These advantages, notwithstanding, the labour-intensive and time-consuming nature of traditional screening presents significant limitations, particularly in the early stages of drug development. On the other hand, computational techniques offer high efficiency, enabling researchers to explore diverse chemical spaces, predict bioactivity and optimize drug leads in record time [141,142]. Computational techniques have facilitated the prediction of pharmacokinetics and pharmacodynamics profiles, including absorption, distribution, metabolism, excretion, and toxicity, which are crucial for assessing the efficacy and safety of therapeutics [143]. Moreover, the application of in silico studies provides insights into the mechanisms of action, binding kinetics and target interactions, enabling identification of chemotypes with improved selectivity and safety profiles. This level of precision is often unattainable through traditional methods alone. Nevertheless, computational predictions must be validated through experimental assays, underscoring the complementary nature of both approaches.

A specific area in NP research utilizing computational studies is data curation and dereplication, which entails the identification and removal of redundant compounds from large databases. Employing this technique has streamlined the NP drug discovery process by reducing the number of compounds that need to be screened for bioactivity. The functionality of NPs has enhanced their ability to modulate most biological targets via multiple interactions [135,144]. However, the synthetic accessibility of these NP compounds is very high due to their complex structure and high molecular weight, rendering them non-druglike [145]. The application of computational strategies has aided in predicting appropriate synthetic routes for the efficient synthesis of complex natural compounds. Additionally, the integration of machine learning techniques and engineering concepts for the development of databases and ontologies has facilitated the annotation and analysis of NP data, further improving outcomes in NP drug discovery research [146]. Overall, NP research has seen significant improvement with the incorporation of computational frontiers. The following section, therefore, reports the advancements made in NP research in the quest to identify effective antiparasitic chemotypes by in silico studies.

Applications of Computational Techniques in Natural Product Research

• Virtual Screening

This is the use of computational strategy in drug discovery to identify from a large chemical library, compounds with the potential to specifically bind to a protein of interest. By employing virtual screening, researchers can screen large libraries of NP compounds against specific biological targets, identifying potential novel compounds with high affinity and selectivity, as well as providing the interactions leading to receptor binding [147,148]. This, in effect, narrows down the vast NP library to a smaller set of promising compounds for experimental validation. Based on the knowledge and availability of the elucidated 3D structure of the target of interest, two forms of virtual screening are known, that is, ligand- and structure-based virtual screening.

Ligand-based virtual screening (LBVS), which explores the structural and chemical properties of known ligands to predict the potential binding affinity of novel compounds, has emerged as an attractive approach in the identification of NPs with antiparasitic bioactivities [149]. In essence, LBVS examines the comparative molecular similarity of compounds with known and unknown activities to build a model which is used to select potential hits from a large chemical library. Associated with LBVS are pharmacophore modelling, molecular similarity, and QSAR, which have facilitated the identification of novel hit compounds mostly from the NPs for the treatment of parasitic diseases and other human ailments [150,151]. Despite not involving the target information, these strategies highlight the power and versatility of LBVS in drug design for identifying new compounds significantly different from the ligands used in generating the models with equal or improved potency.

In the area of NP chemistry for parasitic infections, recent successful cases employ the application of various LBVS strategies to create models for the identification and prediction of biological activities of antiparasitic NP compounds [152,153]. For instance, in the case of sterol methyltransferase (SMT), an enzyme which catalyzes the transfer of methyl group from S-adenosylmethionine to the C24 position of zymosterol, six known inhibitors of L. donovani sterol methyltransferase (LdSMT) were used in generating a robust pharmacophore model, which was then used to screen a library of NPs from Interbioscreen Limited. Compounds with a pharmacophore fit score greater than 50 were then virtually screened against the modelled LdSMT, generating a spiroindoline compound, 160 (Figure 14), with the lowest binding energy of −10.1 kcal/mol compared to the known inhibitor, 22,26-azasterol, with a binding energy = −7.6 kcal/mol [154]. Furthermore, employing quantitative structure–activity relationship (QSAR)-based virtual screening identified two sesquiterpene lactone NPs compounds, LDT-597, 161 and LDT-598, 162 (Figure 14), with potential antimalarial activities [155].

Structure-based virtual screening (SBVS) involves the knowledge of 3D structures of proteins to identify molecules with specificity to bind at the active sites of the protein surface. The advancements made in molecular biology and omics, such as genomics and proteomics, have identified essential biological targets, as well as fundamentally providing analyses of the 3D macromolecular structures of pharmaceutical interest [156,157]. Corroboratively, a thorough understanding of the spatial and energetic parameters underpinning ligand–receptor binding interactions has been made possible by advancements in analytical methods used for structure elucidation and molecular studies, comprising calorimetry, NMR, and X-ray crystallography [158]. Subsequently, public databases such as National Center for Biotechnology Information (NCBI), Uniprot, Protein Data Bank, and Protein Data Bank in Europe have made available primary amino acid sequences or 3D structures of target proteins, which can easily be retrieved. Despite the absence of most validated targets which have the potential to hinder NP research with antiparasitic activities, the availability of robust homology modeling strategies like Alphafolds, I-Tasser, Modeller, Swissmodel and many more has enhanced SBVS. The application of SBVS has already afforded NP compounds with antiparasitic therapeutic potential. Knowledge of Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) as a vital enzyme in the isoprenoid biosynthetic pathway required for growth and survival as an attractive target was explored for designing antimalarials. PfDXR, together with fosmidomycin, was used to generate a pharmacophore model, which was then used to screen a focused NP library of 100,000 compounds, followed by a virtual screening against the target protein. Four compounds, 163 to 166 (Figure 14), with respective binding energies of −12.1, −12.0, −11.3 and −11.1 kcal/mol, were identified as hit compounds showing good ADMET properties [159].

Schistosoma mansoni is one of the causative agents of human schistosomiasis known to be unable to biosynthesize purine nucleotides, making enzymes of the purine salvage pathway an attractive therapeutic target for antischistosomal drug design. High-throughput virtual screening of NPs against the Schistosoma mansoni purine nucleotide phosphorylase (SmPNP) enzyme identified two neolignans, 167 and 168, with the potential to silence SmPNP [160]. Moreover, the absence of vaccines and the drawbacks of the current drugs for leishmaniasis treatment have prompted the scientific community to explore NPs for their antileishmanial activities. Arginase is considered a viable target for drug design against leishmaniasis, as its presence in the polyamine pathway catalyzes the formation of L-ornithine and urea from L-arginine. Employing SBVS from the 2000 NPs and derivatives of the Nuclei of Bioassays, Ecophysiology, and Biosynthesis of Natural Products Database (NuBBE_DB) identified three compounds 169, 170, and 171 (Figure 13) with good binding energies against arginase of L. infantum, L. mexicana and L. brasiliensis [161].

• Molecular Docking and Molecular Dynamics Simulations

Molecular docking is conducted to predict the preferred orientation of a ligand in a protein target, resulting in the formation of a stable complex. Applying this technique enables the preferred orientation of the ligand within the binding pocket of the receptor to be determined. Molecular docking, when explored, can predict the binding energy, as well as the interaction existing between protein and ligand [162,163]. Integration of molecular docking has suggested potential binding sites and interactions critical for the identification of, as well as guiding in the design of, NPs as antiparasitic agents [164,165]. Complementing molecular docking in computational studies to reduce attrition rates in experimental screening, molecular dynamics (MD) simulations play a pivotal role; thus, MD provides insights into the behaviour of the ligands, including NP compounds in biological systems, allowing researchers to predict their binding modes, stability, and interactions with biomolecules. Moreover, in the absence of experimental validation, a combination of MD simulations and Molecular Mechanics Poisson–Boltzmann Surface Area (MM-PBSA) or Molecular mechanics with generalised Born and surface area solvation (MM-GBSA) computations has predicted the free binding energy of the complex formed from the target protein and NPs, which has aided in the discovery of new antiparasitic chemotypes [166,167]. Numerous studies have demonstrated the utility of molecular docking and molecular dynamics simulations in the design of NPs as antiparasitic agents [161,168,169,170]. An example is the application of in silico studies to identify limonoids extracted from the bark of the stem of Entadrophragma angolense as antimalarial agents [171]. Virtual screening of sixteen limonoids against PvFKPB35 also identified two lead compounds 172 and 173 (Figure 15) showing binding energies of −6.3 and −5.4 kcal/mol, respectively, compared to the experimental binder 174 (−6.3 kcal/mol). The compounds were predicted to be druglike, conforming to Lipinski’s rule of five with negligible toxicity issues [171]. By employing a computational study, these limonoids were suggested to be biologically active, setting the platform to use the scaffolds for the design of new antimalarial chemotherapeutics. A similar study identified two compounds 175 and 176 obtained from the African NPs-derived compounds showing binding energies of −9.5 and −9.2 kcal/mol relative to the known inhibitor GSK3186899, 177 (−8.5 kcal/mol) against L. donovani cell division cycle-2-related kinase 12 (CRK12) [172]. The study further suggested that the amino acid residue Lys488 is essential for ligand binding in the ATP pocket of the target protein [172].

Ethnopharmacological research has identified numerous natural sources—particularly medicinal plants—as promising treatments for parasitic infections. While many of these NPs are known to possess antiparasitic activity, the specific bioactive compounds and their molecular targets often remain poorly characterized. However, recent advancements in computational studies like molecular docking and molecular dynamics simulation analysis have significantly contributed to elucidating the binding modes and potential mechanisms of action of these compounds.

Employing molecular docking and molecular dynamics simulations studies on Cecropia obtusifolia (Cecropiaceae), the NPs isoorientin, 178 (−9.1 and −8.8 kcal/mol) and chrysin, 179 (−9.6 kcal/mol) were identified as the best binders of 1CET, 2BL9, and 4ZL4, respectively. MD studies revealed the compounds to show stable complexes with the target proteins, validating the pose from the docking studies [173]. In a similar study, hardwickiic acid, 180, isolated from the stembark of Croton sylvaticus, possessed leishmanicidal activities with binding energies of −8.0, −7.8, −7.6, −7.5, −7.4 and −7.1 kcal/mol, respectively, against targets LmGCL, LmPTR1, LdTR, LmTR, LdGCL, and LdPTR1. MD studies corroborated results from the docking studies, suggesting the amino acid residues Lys16, Ser111, and Arg17 to be critical for ligand binding [174]. In another study, the biological target of inhibition was the focus for some NPs which had been demonstrated experimentally in vitro or in vivo to possess antileishmanial activities. Docking and MM-PBSA computations revealed that betulin, 181, betulinic acid, 182, ismailin, 183, oleanolic acid, 184, pristimerin, 185, and ursolic acid, 186 (Figure 15), inhibit LmPTR1 with binding energies ranging from −11.2 to −5.2 kcal/mol and binding free energies of −87 to −148 kJ/mol [175]. The results suggest the NPs exhibit their mode of action via Leishmania pteridine reductase inhibition.

Further, recent research in NP chemistry has found results from computational studies to be corroborated by experimental evaluations. For instance, by virtually screening a library of 1327 NPs from insects against the thioredoxin glutathione reductase of Schistosoma mansoni, hit compounds were identified to bind at the doorstop pocket of the target protein. Experimental validation of the docking studies was carried out by testing the compounds against adult and juvenile Schistosoma parasites in vitro. The compound buprestin H, 187 (Figure 16), isolated from the jewel beetle was found to be the most potent in suppressing adult and juvenile worms with concentrations lower than 20 and 5 μM, respectively. Testing the compound against the human HepG2 cell line for the cytotoxicity analysis revealed the compounds to be non-toxic to human cells, producing a cytotoxic concentration (CC₅₀) of 55 µM [176]. A novel labdane diterpene, 18α-O-trans-p-feruloyl-15-methyl-8(17)-labdanoate, 188, isolated from the roots of Vachellia nilotica and structurally elucidated via NMR and LC-MS, was found to possess antiparasitic activities with an IC₅₀ of 0.0177 µM against Trypanosoma brucei and 0.0154 µM against Leishmania major. Molecular docking studies predicted that the antileishmanial and antitrypanosomal activities were due to inhibition of cysteine proteases, producing binding energies of −10.5 and −7.8 kcal/mol, respectively [177]. Despite the antitrypanosomal efficacies of extracts and fractions of U. ovata against Trypanosoma brucei brucei GUTat 3.1 with IC₅₀ ranging from 0.12–4.40 μg/mL, the mechanism of binding was unknown [178]. Docking 17 NPs from U. ovata against farnesyl diphosphate synthase (FPPS) and ornithine decarboxylase (ODC) enzymes showed mannosamine, 189, to be the most promising lead, with a binding energy of −6.4 kcal/mol against FPPS [178]. The study identified 189 to exhibit antitrypanosomal activity with FPPS as the target of inhibition [178]. Moreover, employing a combination of in silico and in vitro studies identified sophoraflavanone G, 190 (Figure 16) to be the most potent, exhibiting an IC₅₀ of 19.2 µM against L. major. The inhibition of pteridine reductase is predicted to be the potential mechanism of action according to molecular docking studies [165].

• Repurposing

The overwhelming incidence of drug resistance and high cases of antiparasitic drug inefficiencies call for urgent attention in the era of an ever-increasing rate of parasitic infections globally. The long duration for new drugs to reach the market, coupled with the high attrition rate, paints a gloomy picture for antiparasitic drug discovery. Drug repurposing, also called drug repositioning or re-profiling, offers an alternative use for already existing drugs. Compared to old traditional means of drug discovery, this technique offers a faster and modest means of identifying new therapeutic agents, especially for parasitic infections, which are mostly less attractive. Application of this strategy has afforded the new compounds with antiparasitic activities from drugs meant for different indications. A notable example is doxycycline, 191 (Figure 17), a tetracycline derivative with a broad spectrum of antibiotic potential currently used for the treatment of malaria [179]. Similarly, clindamycin, 192, a lincosamine antibiotic for the treatment of acne, has found use as an antimalarial chemotype [180,181]. To find new therapeutics for combating visceral leishmaniasis afforded miltefosine, 193 and paromomycin, 194 were originally designed for the treatment of breast cancer and antibiotic, respectively. The antitumor agent, elfornithine, 195 (an ornithine decarboxylase enzyme in the polyamine biosynthetic pathway inhibitor), is currently the prescribed drug for treating African sleeping sickness.

Despite the structural and synthetic complexity of NPs, their low cytotoxicity and diverse pharmacological activities render them a better choice in the search for antiparasitic candidates. Consequently, several NPs have been repurposed for the treatment of parasitic diseases. Atovaquone, 196, released in 1992 for the treatment of malaria prophylaxis, has found use in the treatment of toxoplasmosis. A lactol methyl ether antimalarial drug, artemether, 197, is currently used in Schistosoma mansoni prophylaxis. In addition, amphotericin B, 10, isolated from Streptomyces nodosus for the treatment of local mycotic infections and fatal fungal infections, is now one of the few chemotherapeutic options for leishmaniasis treatment.

The application of in silico studies in the current dispensation of drug discovery has further expedited the identification of NPs as antiparasitic agents. By virtually screening retinoic acid, 198 and its derivatives to a homology modelled L. donovani C-24 sterol methyltransferase, 198 showed the least binding of −9.9 kcal/mol compared to the reference compound zymosterol, 199 (−8.5 kcal/mol). The molecular dynamic simulation studies suggest that 40 forms a stable complex with the target protein, and in vitro studies show a twofold reduction in the parasite load after treatment with 198 (Figure 17). The study also revealed a decrease in the level of ergosterol in L. donovani, possibly due to the inhibition of C-24 sterol methyltransferase [182]. Employing similar studies, a molecular docking of the FDA-approved drug library to primase protein gave mupirocin, 200 (Figure 17), recording a binding energy of 10.65 kcal/mol. In vitro evaluation of biological activity showed a 69.1% cell viability compared to 1.10 (100%) [183]. Based on in silico and in vitro studies 200, an NP isolated from the bacterium Pseudomonas fluorescens and originally used as an antibiotic can be repurposed for the treatment of leishmaniasis. The monocyclic sesquiterpene alcohol, α-bisabolol, 201 first isolated from Matricaria chamomilla and is known for its anti-inflammatory and anti-irritant properties, as well as being found in cosmetics to enhance skin texture and appearance [184]. Molecular docking and molecular dynamics simulation studies suggest 201 could be repurposed for the treatment of sleeping sickness, as it was found to interact with critical amino acids (Met115, Tyr112, and Trp23) in the binding domain of T. brucei trypanothione reductase [185]. In a similar study, Berhanu et al. identified ricinoleic acid, 202 from the seed oil of Ricinus communis L. to possess antihelmintic activities. Molecular docking studies suggest the mode of action of 202 is via succinate dehydrogenase inhibition, recording a binding energy of −5.408 kcal/mol [186]. Ethnopharmacological data revealed that 202 (Figure 17), a major fatty acid in castor oil, is useful as a laxative, anti-inflammatory agent, wound healing, and skin conditioning [187].

7. Innovative Approaches to NP-Based Drug Discovery

7.1. Combination Therapies

Drug resistance is the main obstacle in managing many parasitic species, impeding the achievement of clinical outcomes [188]. Parasitic infections significantly affected by drug resistance include malaria, with Southeast Asia being the region most impacted [189,190,191,192], Babesiosis (primarily in the United States) [193], Toxoplasmosis (drug resistance primarily noted in Brazil) [194], Leishmaniasis (primarily in North Bihar, India) [195,196], and Human African trypanosomiasis (which is present in Africa) [196] (Table 1). The concurrent use of two or more drugs does not inherently guarantee that their efficacy will surpass that of single-agent treatments. Drug interactions can be categorized as antagonistic, additive, or synergistic. Antagonism is observed when two drugs impede the therapeutic effects of one another. Additive occurs when two drugs produce a combined effect that matches the predicted outcomes of each drug when administered individually. Synergism arises when the combined effect of the drugs exceeds the anticipated outcomes of their individual effects. Furthermore, an inert agent, which lacks an individual effect, may enhance the action of a secondary drug.

This section presents the role of preclinical drug combinations in identifying lead candidates from multiple initial hits and optimizing procedures for drug scale-up [197]. These efforts encompass laboratory research and animal testing to evaluate investigational new drugs for their potential therapeutic effects [197,198]. Additionally, preclinical studies assess drug delivery systems using cellular and animal models to determine pharmacokinetics, biodistribution, and toxicity profiles [199,200]. In contrast, clinical drug combinations involve the transition from the laboratory and animal models to humans. These studies evaluate the safety, efficacy, and tolerability of drug combinations across diverse range patient cohorts, thereby reflecting real-world therapeutic applications [201].

Table 1. Parasitic infections, combination therapies, and most affected geological regions.

Parasitic Infection	Combination Therapy	Geological Regions Mostly Affected by Drug Resistance	References
Malaria	a 1, b 2, c 3	Southeast Asia	[189,190,191]
Babesiosis	d 4	United States	[193]
Toxoplasmosis	e 5, f 6	Brazil	[194]
Leishmaniasis	g 7, h 8, i 9	North Bihar in India	[195,196,202]
Human African Trypanosomiasis	j 10, k 11, l 12	Africa	[203,204,205,206]

¹ B02 and artemisinin, 23 (a natural compound from the Artemisia annua plant). ² Prochlorperazine, 203 (Figure 18) and Chloroquine, 24 (derived from the natural pharmacophore of quinine, 22). ³ Chlorpheniramine, 204 + Mefloquine, 205. ⁴ Diminazene aceturate, 9 and Clofazamine, 206 (inspired by lichen-derived natural diploicin). ⁵ Clindamycin, 207 (derived from natural lincomycin) and azithromycin, 208 (derived from the natural pharmacophore of erythromycin). ⁶ Simvastatin, 209 (derived from natural lovastatin) and pyrimethamine, 14. ⁷ Tamoxifen, 210 + amphotericin B, 10 (natural product). ⁸ Lovastatin, 211 (NP) + chromium chloride, 212. ⁹ Nelfinavir, 213 + amphotericin B, 10 (NP) [202]. ¹⁰ Chlamydomonas rheinhardtii extract + nifurtimox, 214 [204]. ¹¹ Amiodarone, 215 (naturally inspired compound) + benznidazole, 216 [205]. ¹² Limonene, 217 and citral, 218 (Figure 18) of Lipia alba [206].

Table 1. Parasitic infections, combination therapies, and most affected geological regions.

Parasitic Infection	Combination Therapy	Geological Regions Mostly Affected by Drug Resistance	References
Malaria	a 1, b 2, c 3	Southeast Asia	[189,190,191]
Babesiosis	d 4	United States	[193]
Toxoplasmosis	e 5, f 6	Brazil	[194]
Leishmaniasis	g 7, h 8, i 9	North Bihar in India	[195,196,202]
Human African Trypanosomiasis	j 10, k 11, l 12	Africa	[203,204,205,206]

¹ B02 and artemisinin, 23 (a natural compound from the Artemisia annua plant). ² Prochlorperazine, 203 (Figure 18) and Chloroquine, 24 (derived from the natural pharmacophore of quinine, 22). ³ Chlorpheniramine, 204 + Mefloquine, 205. ⁴ Diminazene aceturate, 9 and Clofazamine, 206 (inspired by lichen-derived natural diploicin). ⁵ Clindamycin, 207 (derived from natural lincomycin) and azithromycin, 208 (derived from the natural pharmacophore of erythromycin). ⁶ Simvastatin, 209 (derived from natural lovastatin) and pyrimethamine, 14. ⁷ Tamoxifen, 210 + amphotericin B, 10 (natural product). ⁸ Lovastatin, 211 (NP) + chromium chloride, 212. ⁹ Nelfinavir, 213 + amphotericin B, 10 (NP) [202]. ¹⁰ Chlamydomonas rheinhardtii extract + nifurtimox, 214 [204]. ¹¹ Amiodarone, 215 (naturally inspired compound) + benznidazole, 216 [205]. ¹² Limonene, 217 and citral, 218 (Figure 18) of Lipia alba [206].

Figure 18. Chemical structures of natural compounds (or derivatives) used in combination with synthetic medications.

Figure 18. Chemical structures of natural compounds (or derivatives) used in combination with synthetic medications.

7.1.1. Combination Therapy for Malaria

The global expansion of malaria prevention interventions and the development of new technologies have saved millions of lives and reduced malaria mortality by 36% between 2010 and 2020, fostering hopes and plans for elimination and eventual eradication [190]. However, recent years have stagnated progress, underscoring the necessity for ongoing vigilance and research [190]. Combination therapy is among the most effective methods currently employed to treat malaria, and it involves the use of two or more therapeutic agents together. The gold standard for first-line treatment of uncomplicated falciparum malaria remains ACTs [190]. Two notable combinations utilized in malaria treatment include artemether, 197 (a natural pharmacophore) (Figure 17) with lumefantrine, 219, and dihydroartemisinin, 220 (a natural pharmacophore) combined with Piperaquine, 221 (Figure 18).

In malaria management, combining quinine, 22 (Figure 2), with quinidine, 222 and cinchonine, 223 shows 2–10 times greater effectiveness in vitro against quinine-resistant strains. Furthermore, this alkaloid mixture demonstrates a more reliable effect compared to each alkaloid used individually. Although this combination has been applied clinically, there is no published research examining whether a synergistic effect also occurs in human patients [190,207].

Combination therapy with 23 (Figure 2) and 132 is beneficial because 132 directly fights malaria, enhances 23’s effects, and works well with other anti-malarial plants. It inhibits cytochrome enzymes, potentially extending the half-life of anti-malarial drugs and reducing multidrug resistance. In animal studies, 132 demonstrates immunomodulatory effects, protects against cerebral malaria, and is tolerated in high doses. Turmeric and black pepper, often used as spices, are readily available, making their combinations suitable for clinical trials. Ginger could also be an effective adjunct due to its anti-emetic properties.

223 (from Cinchona bark) is a promising candidate for clinical trials, offering several mechanisms of action. It has direct anti-malarial activity, reverses resistance, and likely has fewer side effects than 22. Whole Cinchona bark extracts were clinically safe and effective for treating uncomplicated falciparum and vivax malaria in extensive 1930s trials [207]. While 223 (Figure 18) combined with 22 is safe and effective, it has not been assessed for drug-resistant malaria treatment [208].

Further individual and collective research efforts are essential. There is a lack of clinical studies on pharmacokinetic synergy and immunomodulation. More investigations are needed into pharmacodynamic synergy, resistance reversal, and reducing side effects. This may include trials of pure compound combinations (like 23 + 131 + 132) and herbal remedy mixes (like Artemisia annua leaves + Curcuma longa root + Piper nigrum seeds). The first could enhance existing pharmaceuticals, while the latter might boost herbal remedies in areas lacking modern drugs [207].

7.1.2. Combination Therapy for Babesiosis

Diminazene aceturate, 9 (Figure 1), is the first-line drug for Babesia gibsoni-infected dogs in Japan [209]. In treating Babesia gibsoni, combining 9 with vincristine sulfate, 224, emetine dihydrochloride, 225, harringtonine, 226, or cephaeline·2HBr, 227 (Figure 19) shows synergistic effects, potentially lowering drug doses and side effects. However, these combinations have a narrow safety margin and may cause severe adverse effects. The half maximal inhibitory concentrations (IC₅₀) were determined for each compound, and synergy was assessed using combination index (CI) values. Results showed that 224 + 9, 225 + 9, 226 + 9, and 227 + 9 were synergistic, while lycorine, 228 + 9, was antagonistic [210].

7.1.3. Toxoplasmosis

The standard treatment for toxoplasmosis involves a combination of pyrimethamine, 14 and sulfadiazine, 15 (Figure 1), along with folinic acid, 229 (Figure 19) to counteract bone marrow suppression. This regimen is mainly used for HIV-infected individuals and cases of congenital or ocular toxoplasmosis [211]. The synergistic action of 14 and 15 disrupts folic acid synthesis in the parasite, but the therapy can also harm human cells [212]. For patients allergic to 15, 14 is paired with clindamycin, 230, which inhibits protein synthesis but has limited penetration into the cerebrospinal fluid. Other alternatives include combining 14 with macrolides like clarithromycin, 231, roxithromycin, 232 (Figure 19), or 208, which also target protein synthesis. While effective, these treatments may cause various adverse effects [37]. A novel combination therapy for toxoplasmosis involving 14 and Nigella sativa seed oil extract (NSO), rich in thymoquinone, 233, was evaluated for its effectiveness [213]. While NSO alone showed no significant anti-parasitic activity, it strongly stimulated immune responses, particularly increasing IFN-γ levels. When combined with 14, NSO significantly improved outcomes by delaying mortality, reducing parasite load, and enhancing tissue pathology in infected mice. This combination performed comparably to the established 14 and 230 (Figure 19) therapy, suggesting that NSO’s immunostimulatory properties can enhance the efficacy of traditional treatments [213].

7.1.4. Leishmaniasis

Withaferin A, 234 (Figure 19), exhibits antileishmanial activity primarily by inhibiting the Pteridine reductase 1 enzyme in Leishmania donovani, and its combination with existing antileishmanial drugs has shown promise in disease management [214]. Additionally, a study investigating triclabendazole, 235—typically used for fascioliasis—combined with amphotericin B, 10 (Figure 1) against L. amazonensis revealed a synergistic effect. This combination caused morphological changes in the parasite and significantly lowered the IC₅₀ values by 3 to 120-fold, compared to either drug used alone, indicating enhanced efficacy against intracellular amastigotes [215].

7.1.5. Human African Trypanosomiasis

Chemotherapy regimens such as the nifurtimox, 214 (Figure 18)–eflornithine 195 (Figure 17) combination therapy (NECT) have proven to be highly effective in treating trypanosomiasis and represent a significant advancement in combination treatment strategies. 195, a chemically modified derivative of ornithine, 236 (Figure 19), plays a central role in this therapy. NECT has demonstrated strong clinical success and remains a cornerstone in current anti-trypanosomal treatment protocols. Its effectiveness also highlights the potential for developing orally administered alternatives, underscoring the need for continued research and innovation in combination chemotherapy for parasitic diseases [215,216].

7.1.6. Other Parasitic Infections

Ivermectin, 3 (A and B) (Figure 1), remains the cornerstone of community-directed treatment for onchocerciasis. For the global control of lymphatic filariasis, a combination of diethylcarbamazine, 237 (Figure 19) or 3 with albendazole, 238 is recommended, with the standard treatment for infected individuals being 238 plus 3. In the case of schistosomiasis, three primary drugs are used: metrifonate, 239, oxamniquine, 240 (Figure 19) and praziquantel, 6 (Figure 1). Notably, 240 is produced through microbial hydroxylation of its synthetic precursor using Aspergillus sclerotiorum [217].

Overall, combination therapies play a critical role in managing parasitic infections, especially in the face of rising drug resistance. They not only enhance treatment efficacy but also help extend the utility of existing approved medications.

7.1.7. Pharmacokinetic and Pharmacodynamic of Combinational Therapies

Pharmacokinetic (PK) and pharmacodynamics (PD) interactions of NPs as combination therapy for parasitic infections offer both beneficial and detrimental effects. PK interactions which encompass the processes of drug absorption, distribution, metabolism, and excretion, can influence the activity of drug transporters or metabolic enzymes [218]. NPs with the potential to inhibit cytochrome P450 (CYP) enzymes or drug transporters like P-glycoprotein can alter the plasma concentrations of co-administered agents and potentially impact their efficacy [219,220]. Moreover, some NPs demonstrate gut wall permeability alterations and may hinder intestinal absorptions [220,221]. Despite the synergistic effects of combination chemotherapy of antiparasitic NPs, PK interactions can lead to increased or decreased drug levels, potentially causing therapeutic failure. PD interactions, on the other hand, occur at the level of drug’s mechanism of action, enhancing therapeutic outcomes through synergism or reducing efficacy through antagonism [218,222]. In synergistic PD interactions, the combined effects of individual compounds may surpass the sum of their individual effects, whereas in the case of antagonism, one compound may interfere with the action of another, reducing the overall antiparasitic efficacy of the combined agent [218,223]. Due to these complexities, careful selection and evaluation of NPs for use in antiparasitic combination therapies are essential to maximize therapeutic efficacy while minimizing adverse interactions.

7.2. Nanotechnology: Enhancing the Delivery and Efficacy of Natural Products

Globally, parasitic diseases continue to pose a significant threat, especially in poverty-stricken areas [224,225]. While NPs have demonstrated promising therapeutic potential as antiparasitic agents due to their diverse bioactive compounds, their clinical utility is often hindered by poor solubility, limited bioavailability, stability issues and inadequate specific target delivery [226]. These pharmacological limitations are relatively characteristic of conventional formulations. Therefore, while cost-effective and relatively simple to manufacture, conventional systems often fail to maintain therapeutic concentrations at the site of infection, reducing overall efficacy and increasing the risk of treatment failure [227,228]. Advances in nanotechnology have, however, provided innovative means of overcoming these challenges and improving efficacy while reducing side effects of NPs as antiparasitic candidates [229,230]. Liposomes, one of the many nanocarrier systems used in nanotechnology, are phospholipid vesicles required to encapsulate both hydrophilic and hydrophobic compounds, including NPs. Their adaptability and biocompatibility make them ideal carriers for complex NPs as antiparasitic chemotypes [231]. The application of this technique has afforded amphotericin B liposomes composed of hydrogenated soy phosphatidylcholine, 241, cholesterol, 242, and distearoyl phosphatidylglycerol, 243 (Figure 20). By targeting infected macrophages through phagocytosis, amphotericin B liposomes are used to treat leishmaniasis [232]. Artemisinin-based liposomes consisting of 241, 242, and PEGylated lipids are also used for combating malaria [233]. Similarly, benznidazole, 216 (241 and phosphatidylserine, 244) and praziquantel, 6 (241 and 242) liposomes are used for treating Chagas and schistosomiasis, respectively [234,235].

Another class of nanocarrier with significant potential due to the distinct merits in terms of biodegradability, biocompatibility, and controlled release properties is polymeric nanoparticles. The chitosan-based system is a natural polymeric nanoparticle with excellent mucoadhesive properties and enhanced permeability for effective oral delivery of hydrophobic NPs. Obtained from chitin, this nanoparticle possesses improved stability and bioavailability and is applied in chloroquine delivery against Plasmodium berghei infection [236]. The alginate is also a natural polymeric nanoparticle obtained from marine sources with massive encapsulation properties. By forming stable matrices, this nanoparticle offers protection for sensitive compounds from degradation. The NPs spiramycin, 245 (Figure 20) and propolis are critical for the treatment of toxoplasmosis, are delivered by alginate nanoparticles [237]. Other FDA-approved synthetic nanoparticles, such as poly(lactic-co-glycolic acid) and polyethylene glycol, have also been used in the delivery of NPs, including 23 and 132 for the treatment of malaria and other parasitic diseases [238,239,240].

Aside from their antiparasitic activities, green-synthesised metal nanoparticles, particularly gold and silver, possess delivery capabilities for NPs [241]. This dual approach has made metal nanoparticles a sought-after nanocarrier for NPs necessary for the treatment of various parasitic infections [241,242]. Despite the magnetic and mechanical properties as well as the melting point and surface area enhancing the pharmacological profiles of these nanometals, the unsustainable and toxic reagents for their synthesis have hindered their use for the treatment of various diseases, including parasitic infections [243,244]. However, the recent interest in green synthesis, which explores plant extracts in an easy and eco-friendly process to produce nanoparticles for their antiparasitic activities, has been ignited [245]. Different studies have therefore shown that NP metal nanoparticles displayed high activities for the treatment of parasitic diseases [246,247,248]. By employing aqueous extract of Rhazya stricta as a source of reducing and stabilizing agents, Ahmad and co-workers in 2017 synthesized gold nanoparticles, which demonstrated effective activity in suppressing the growth of intra-THP-1 amastigotes with an IC₅₀ of 43 μg/mL [248]. Interestingly, no cytotoxicity was observed when THP-1 cells were exposed to these nanoparticles for 24 h [248]. A similar study employed Olax nana Wall. ex Benth aqueous extract in the synthesis of gold and silver nanoparticles and evaluated for biocompatibility and antileishmanial activities. Without exhibiting any cytotoxicity against the newly isolated human macrophages, the biogenic gold-silver nanoparticles showed profound activity against Leishmania tropica (KMH23) promastigotes (IC₅₀ = 12.56 and 21.52 μg/mL) and amastigotes (17.44 and 42.20 μg/mL) [249]. The use C. dentata leaf extract afforded the green synthesis of selenium nanoparticles which at LC₅₀ of 240.714 mg/L demonstrated significant larvicidal activity against An. stephensi [250]. Altogether, a few plant extracts have been employed in the green synthesis of various nanometals, including zinc, iron, copper, titanium, and nickel, for their antiparasitic activities [251,252,253].

Overall, nanotechnology offers superior advantages over conventional formulations in delivering specific target capabilities, enhancing drug stabilities, overcoming parasite resistance mechanisms, reducing systemic toxicities, and enabling multiple targets activities [254,255]. Despite these advantages, nanotechnology is not without limitations. Long-term safety data and human clinical trials remain limited. Toxicological profiling using in vitro and in vivo models is essential before widespread clinical adoption. Additionally, complex formulation procedures, higher production costs, regulatory hurdles, scale-up and reproducibility remain critical issues that need to be addressed [256].

7.3. Genetic Engineering

Development and current progress in parasite genomics have facilitated a deeper understanding of the genetic structures of parasites, including various species of Plasmodium, Toxoplasma, Leishmania, and Trypanosoma. Limitations on optimal drug performance and commercial viability have posed challenges stemming from limited production from natural sources, sub-optimal pharmacokinetic and pharmacodynamic properties, as well as drug resistance to current therapies [194]. The attempt to solve this challenge considers the use of combination therapy, which is often hindered by limited access to production platforms, technologies, and various compounds (synthetic/semi-synthetic/natural) for testing [257,258,259]. Innovative next-generation sequencing (NGS) methods, including high-throughput sequencing, have identified critical genes associated with metabolic pathways, drug resistance, and life cycle adaptations. Developments in genomics have allowed for an in-depth exploration of biological processes and the evolutionary adaptations of parasites [216]. With knowledge of the genome sequences of naturally sourced antiparasitic agents that produce complex drug molecules, synthetic biology and genetic engineering can pave the way to obtaining effective molecules with fewer obstacles. Genetic engineering is a critical field in biosynthesis as it connects alterations made in genes with the morphed molecule isolated. Technologies utilized in genetic engineering include CRISPR-Cas9 technology, gene cloning, REDIRECT PCR-targeting or recombineering [50,260,261,262].

Genetic engineering plays a pivotal role in enhancing the potency and yield of antiparasitic NPs, particularly when chemical synthesis is challenging or requires harsh conditions. By enabling enzymatic modifications, it facilitates the biosynthesis of potent compounds directly from microbial or plant hosts. A key strategy involves identifying the core biosynthetic gene clusters responsible for producing bioactive compounds. Once identified, metabolic flux can be redirected toward the desired product by knocking out competing pathways that consume shared precursors or intermediates. This targeted gene disruption reduces metabolic competition and enhances the yield of bioactive analogues. Moreover, genetic tools allow for the activation of silent or repressed genes within these clusters. Bioinformatic analyses can reveal such genes, which may be activated by inserting strong constitutive or inducible promoters. Strategic promoter placement, especially upstream of bottleneck enzymes like inactive tailoring enzymes, ensures efficient conversion of intermediates into the final bioactive product.

In cases where native producers are difficult to culture or genetically manipulate, heterologous expression offers a powerful alternative. Essential gene clusters can be cloned into more tractable hosts—such as Escherichia coli—which offer advantages like ease of cultivation, higher protein expression, and improved control over production rates. For instance, the heterologous expression of the gene cluster responsible for ikarugamycin, an antibiotic with antiprotozoal activity, in E. coli has enabled more efficient production of this valuable compound [215].

Post-engineering challenges often arise due to host-pathway incompatibilities, where newly introduced biosynthetic routes or enzymes may exhibit instability or reduced functionality in heterologous systems. Additionally, structural modifications introduced through genetic engineering can lead to unpredictable structure–activity relationships, particularly when the engineered components are not well-integrated with the host’s native metabolic framework.

8. Clinical Trials in NP Research

NPs have historically served as a crucial source in drug discovery, with approximately 50% of all approved pharmaceuticals from 1981 to 2019 deriving from unmodified NPs, their derivatives, or synthetic compounds incorporating NP-like pharmacophores [263]. For a drug to successfully progress through clinical trials, it must demonstrate both safety and therapeutic efficacy. The primary reason for failure among clinical candidates is insufficient efficacy, even when toxicity profiles are within acceptable limits. These shortcomings are often attributed to inadequate drug-like properties, suboptimal target selection, and other mechanistic or strategic deficiencies [264]. Over the years, several NPs have demonstrated antiparasitic potential and drug-like properties [265]. However, a significant portion of published research focuses on antiparasitic activities using crude extracts or fractions from various natural matrices, with relatively little attention paid to isolated compounds. There are relatively few reliable preclinical studies employing animal models or clinical trials involving human volunteers, and even reports addressing natural antiparasitic substances primarily rely on the outcomes of indirect in vitro antiparasitic tests or evaluations on live parasites. Although some promising compounds with antiparasitic potential and drug-like qualities have been identified, further research is required before any of these molecules can be developed into contemporary antiparasitic medications [34]. The most recent and advanced preclinical and clinical research investigating NPs as antiparasitic medicines is discussed below.

8.1. Artemisinin Derivatives

Artemisinin and its derivatives represent some of the most effective antimalarial agents. By 2019, their global application in malaria treatment had resulted in a 30% reduction in cases and a 37% decrease in malaria-related mortality [3]. Since its discovery, artemisinin has consistently demonstrated therapeutic efficacy against malaria in numerous clinical trials, and ACTs have become integral to modern malaria treatment worldwide [3,266]. The semisynthetic derivatives of artemisinin, such as artemether, artesunate, and arteether, have applications beyond malaria. These compounds exhibit significant anti-helminthic and schistosomicidal properties, proving particularly effective in murine models against parasites including Fasciola, Toxoplasma gondii, Schistosoma malayensis, S. haematobium, S. mansoni, S. japonicum, S. mekongi, and S. intercalatum [266,267,268]. Historically, praziquantel has been the primary treatment for these parasitic diseases; however, its inability to eliminate developing schistosomula leads to reinfections and treatment failures, and monotherapy can promote resistance. Consequently, there is a critical need for novel therapeutics. Clinical investigations have confirmed the efficacy, safety, and cost-effectiveness of artemisinin and its derivatives against S. japonicum [269,270]. Regarding safety, a phase I clinical trial reported no adverse effects associated with repeated oral administration of artemisinin and its derivatives. Numerous trials have validated the safety of a single 6 mg/kg dose of artemether, artesunate, or artemisinin [271]. Most research suggests that artemether is beneficial in the early treatment stages of acute schistosomiasis, as it can reduce infection rates and disease severity [269,270,271,272,273,274,275]. Nonetheless, some studies indicate that artemisinin derivatives alone do not significantly enhance clinical outcomes [276]. Furthermore, artemisinin and its derivatives have demonstrated utility in schistosomiasis prevention. Lin et al. reported a preventative effect with a single 6 mg/kg dose administered every 15 days [277]. These findings have been corroborated by subsequent studies conducted among populations residing in schistosomiasis-endemic regions [271,273,274,278,279,280].

Evidence indicates that prolonged use of artesunate for the treatment of S. japonicum has led to decreased sensitivity [281,282]. Furthermore, research suggests that artemisinin and its derivatives may also inhibit Fasciola hepatica. For instance, a randomized controlled trial conducted on human fascioliasis in central Vietnam demonstrated the efficacy of artemisinin and its derivatives against the disease [283]. Although this finding was corroborated by subsequent clinical trial, it concluded that the therapeutic benefit of artemisinin and its variants was insufficient to replace triclabendazole, the conventional drug used for treating fascioliasis [284]. Considerable debate persists regarding the efficacy of ACTs for parasitic infections, despite substantial clinical data supporting their use. Consequently, further phase II/III clinical trials are necessary to more comprehensively establish the safety and efficacy of the antiparasitic properties of artemisinin and its derivatives [285].

8.2. Curcumin for Trichomonosis

Trichomonosis is a protozoan infection caused by the parasite Trichomonas vaginalis. Globally, it represents the most prevalent curable and treatable sexually transmitted infection, with approximately 156 million new cases reported annually among individuals aged 15 to 49 years [286]. The standard treatment involves antibiotics such as metronidazole or tinidazole. However, the emergence of resistance to metronidazole in vaginal infections necessitates the exploration of alternative therapeutic agents, including NPs [287]. In a 2014 in vitro study conducted by Wachter et al., curcumin, a derivative of Curcuma longa, was assessed for its efficacy against three strains of T. vaginalis. The study demonstrated complete elimination of trichomonal cells within 24 h at a concentration of 400 µg/mL of curcumin, with human mucosa exhibiting tolerance at a 50-fold higher dose [288]. These in vitro findings indicate 100% eradication at 400 µg/mL, even against metronidazole-resistant strains. Consequently, curcumin emerges as a promising topical treatment for trichomonosis, particularly in cases of metronidazole resistance [288]. Furthermore, a double-blind randomized clinical trial conducted in 2021 involving 100 married women aged 18–49 evaluated the efficacy of curcumin compared to conventional metronidazole for the treatment of bacterial vaginosis. The results indicated that curcumin was equally effective as metronidazole in treating bacterial vaginosis, with superior efficacy and fewer side effects in alleviating vaginosis symptoms [289]. Therefore, curcumin may be administered alone or in conjunction with other bacterial vaginosis treatments to enhance women’s health. Despite its promising potential, further advanced human trials are warranted, as none have been conducted to date.

8.3. Curcuma Longa + Camellia Sinensis in Livestock

Helminthiasis is a disease that significantly impacts social and economic conditions globally. Among the gastrointestinal nematodes commonly found in domestic animals are Trichostrongylus spp., Teladorsagia circumcincta, Haemonchus contortus, Cooperia spp., Oesophagostomum spp., Chabertia ovina, Bunostomum, Trigonocephalum, and Nematodirus spp. [290]. Anthelmintic drugs, including imidazothiazoles/tetrahydropyrimidines, benzimidazoles, and other macrocyclic lactones, are extensively utilized to effectively control these helminths. However, the frequent application of anthelmintic drugs to manage these livestock nematodes has led to significant drug resistance issues worldwide [291]. Consequently, herbal remedies, particularly NPs derived from endemic plants, are needed as alternatives to synthetic anthelmintics [292]. Ethanolic extracts of C. longa and C. sinensis have been evaluated for their anthelmintic potential against the gastrointestinal nematodes, Trichuris sp., in sheep. The study indicates that a combination of these extracts demonstrated significant ovicidal anthelmintic activity against Trichuris sp. [293]. These findings suggest that these plant extracts could be utilized as a holistic approach to animal health.

8.4. Safety Profiles and Adverse Effects of Natural Products

Natural products are generally widely accepted in traditional medicine use due to their perceived safety; however, emerging evidence suggests that this perception may be overly simplistic. Heydari et al. (2022) posited that both intrinsic and extrinsic toxicities pose significant concerns. Intrinsic toxicity arises from the complex mixtures of bioactive compounds within a single plant, which may interact synergistically or antagonistically, complicating safety evaluations. Extrinsic toxicity includes contamination with heavy metals, misidentification of plant species, and inappropriate clinical use [294].

As noted earlier in the introduction to clinical trials in NP research, the focus of research published on antiparasitic activities has centred on crude extracts or fractions rather than on isolated compounds. A recent comprehensive systematic review and meta-analysis evaluated 162 studies on the antiparasitic efficacy of medicinal plants against gastrointestinal parasites. The review identified 507 plant species from 126 families, with 91 species and 34 compounds demonstrating significant in vitro efficacy [295]. While the review yielded promising outcomes, it also revealed a critical gap: only 57 of the evaluated plants underwent toxicity screening prior to efficacy testing. This finding underscores the need for more rigorous and systematic safety assessments in NP-based drug development

Moreover, the absence of harmonized regulatory frameworks and pharmacovigilance systems for herbal medicines contributes to inconsistent safety reporting. Adverse events associated with NPs are often underreported due to limitations in conventional monitoring systems and the unregulated nature of many herbal formulations.

9. Challenges and Future Directions in NP Research

The integration of NPs into drug discovery modern drug discovery pipelines is presents numerous scientific and operational challenges. Their complex structures and low abundance render their isolation and characterization labour-intensive and time-consuming. Conventional extraction and purification techniques often result in low yields of active compounds or re-isolation of known compounds, limiting their scalability and reproducibility [296]. In certain instances, there are hurdles associated with consistent supply and sustainability for bioactive NPs derived from rare or slow-growing species. This situation is compounded by overharvesting of medicinal plants, particularly in biodiversity-rich tropical regions, threatening both environmental balance and long-term drug development efforts. While NPs often exhibit potent biological activity, many suffer from poor pharmacokinetic profiles, including low solubility, limited bioavailability, and rapid metabolism. There also exists the potential for off-target effects and toxicity remains a significant barrier to clinical translation [297].

To effectively address the multifaceted challenges inherent in NP-based drug discovery and fully realize their therapeutic potential, current and future research efforts must adopt a comprehensive and integrative approach. Key strategic directions include the advancement of extraction and analytical methodologies to improve the efficiency and precision of compound isolation and characterization. The integration of artificial intelligence and machine learning is also critical, offering powerful tools for virtual screening, dereplication, and predictive modeling of pharmacokinetic and toxicological profiles.

Equally important is the promotion of sustainable bioprospecting and cultivation practices to ensure ethical and environmentally responsible sourcing of bioactive compounds. In parallel, expanding the rational design of novel chemical entities inspired by NP scaffolds—such as hybrid molecules, macrocycles, and privileged structures—can enhance biological activity while optimizing drug-like properties. Additionally, fostering multidisciplinary collaboration and the development of open-access databases will facilitate data sharing, accelerate innovation, and minimize duplication of research efforts. In the subsections below, we elaborate on polypharmacy and potential innovations in future research directions.

9.1. Polypharmacology

The complex life cycle of many disease-causing parasites enables them to adapt to a hostile environment and outwit host defences. Consequently, the current one-drug-one-target antiparasitic agents have become inefficient and redundant due to high parasitic resistance [298,299]. Even more alarming is the synergistic resistance associated with most combination chemotherapy for the treatment of parasitic infections [300,301,302]. The clarion call for the identification of chemotypes with the potential to modulate two or more viable biological targets implicated in the pathogenesis of parasitic diseases is timely. Polypharmacology, the use of a single drug molecule to simultaneously and specifically interact with multiple targets, has been employed in the treatment of complex diseases [303,304]. Despite their inherent promiscuity and the associated risk of off-target effects, multi-target drugs are rationally designed to selectively modulate key protein targets, thereby enhancing therapeutic efficacy and mitigating the development of resistance [305,306]. The application of polypharmacology has afforded aspirin, 246 (Figure 21), as an effective analgesic, antipyretic, and anti-inflammatory agent through its dual inhibition of cyclooxygenase enzymes COX-1 and COX-2 [307,308]. The complex nature of cancer has also benefited from polypharmacological strategies. For example, the antineoplastic agent, sorafenib, 247, targets various kinases including platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) receptor tyrosine kinases [309]. Similarly, compound 132 (Figure 11), a bioactive constituent of turmeric, exhibits anticancer activity by modulating different cell signalling pathways implicated in cancer progression, such as NF-κB, AP-1, and STAT3 [310].

Given the proven efficacy of NPs, identifying those that target multiple biological pathways offers a promising approach to overcoming resistance and combating parasitic infections. This can be achieved by evaluating NPs known for their polypharmacology, such as flavonoids, alkaloids, and terpenes, using phenotypic assays on essential pathways like energy metabolism, protein synthesis, and redox balance. The compounds that silence two or more of the biological targets can be subjected to further investigations, including cytotoxicity. In addition, compounds with metal centres such as gold, platinum, ruthenium, iron, and vanadium have shown significant antiparasitic activities. The incorporation of metal centres into NPs facilitates interactions with multiple biological targets, including DNA, redox activity, enzyme inhibition, liberation of reactive oxygen species, and disruption of key metabolic pathways in parasites. By coordinating NPs with metals capable of targeting these critical biological processes, it is possible to design multifunctional NP metal complexes. Applications of metal-coordinated NPs have already been explored in the treatment of various cancers [311]. A coordination complex formed between (−)-epigallocatechin gallate, 89, and molybdenum ions has demonstrated anticancer activity, with the potential to disrupt both endosomal and plasma membranes. Zhen et al. further demonstrated that the complex triggers pyroptosis and anti-tumor immunological responses, enabling broad suppression of different forms of tumors [312]. These complexes hold promise as broad-spectrum therapeutic agents—or potential panaceas—against parasitic diseases by simultaneously targeting multiple essential biological pathways.

Application of computer-aided drug design, such as pharmacophore-based virtual screening, can also be employed in identifying multi-target NPs with antiparasitic activities. This can be achieved by generating a pharmacophore model from moieties of antiparasitic drugs targeting different pathways. The pharmacophore model can be used to virtually screen a curated NP library to identify hits with similar chemical descriptors responsible for the biological activities. Since the pharmacophore model was obtained from drugs targeting different therapeutic receptors, it is expected that the NPs hits may possess the potential to silence multiple biological targets. Further investigations via in vitro and in vivo evaluations can be carried out to confirm the multi-target antiparasitic activities.

9.2. Potential Innovations

Shifting antiparasitic drug development toward target-based design, driven by breakthroughs in genomics and proteomics, provides an alternative means of combating parasitic infections. Unlike traditional approaches that began with identifying a lead compound followed by determining its biological target, modern strategies prioritize identifying molecular targets associated with parasitic infections first and then screening for compounds that can effectively bind to and inhibit these targets. This paradigm shift is particularly urgent given the accelerating spread of parasitic diseases, fueled by factors such as global population migration, environmental degradation, and climate change. Compounding the challenge is the growing resistance to existing antiparasitic therapies, underscoring the critical need for novel treatments [313].

Target-based drug discovery has become more efficient and less resource-intensive, offering improved predictive capabilities for evaluating a compound’s therapeutic potential. One of the earliest and most explored targets is the parasite’s gene-expression machinery, accessible through sequencing technologies. Additional promising targets include cytoskeletal proteins, intracellular signalling molecules, membrane-associated proteins, and enzymes involved in intermediary metabolism. The efficacy of these targets often depends on the parasite’s metabolic state, which can vary under aerobic or anaerobic conditions [314].

Despite these technological advancements, target-based approaches supported by protozoan parasite genomics and proteomics have yet to yield a successful antiparasitic drug. This highlights the complexity of parasitic biology and the need for continued innovation and investment in this critical area of global health.

10. Conclusions

Parasitic diseases remain a major global health concern, particularly in low-resource settings where access to effective treatments is limited. While current therapies have made significant strides, challenges such as drug resistance, limited efficacy, and reinfection persist. NPs, with their structural diversity and unique bioactivities, offer a promising alternative to conventional treatments. Their ability to target critical parasite-specific pathways makes them strong candidates for the next-generation antiparasitic agents.

Advancements in analytical techniques, omics technologies, and computational tools have accelerated the discovery and optimization of NPs. Furthermore, innovative strategies such as combination therapies, nanotechnology, and genetic engineering are enhancing the efficacy and delivery of these agents. Clinical trials of NP-based treatments, including artemisinin and curcumin derivatives, underscore their translational potential.

To fully realize the potential of NPs, a multidisciplinary approach is essential—one that bridges traditional knowledge systems with cutting-edge science. Collaborative efforts between academia, industry, and public health institutions will be critical in translating promising compounds into accessible, affordable, and effective therapies. With sustained investment and innovation, NPs could redefine the therapeutic landscape for parasitic diseases and contribute meaningfully to global health equity.