Although scientific evidence indicates that the safety and efficacy of herbal remedies are limited, herbal drugs are being consumed due to their low undesirable side effects and toxicity, as well as their low price [
42,
43]. Furthermore, in the case of low effectiveness of conventional drugs, there is an increased usage of traditional herbal remedies [
41,
44,
45]. Over last decade, many studies have investigated the effects of medicinal plants on protozoa [
10,
44,
46‐
48] and helminths [
49‐
52].
T. gondii is an opportunistic and life-threatening parasite, particularly in AIDS patients, cancer patients, and organ transplant recipients [
3,
7]; therefore, treatment of human toxoplasmosis seems to be an urgent need for susceptible groups. Several studies have evaluated the effects of herbal medicines on
T. gondii [
10,
36,
53‐
56].
A. indica (Neem) belongs to Mahogany family and has been used as herbal medicine, particularly in Africa and south of Asia [
57]. The Neem extracts have exhibited immunomodulatory, anti-hyperglycemic, anti-carcinogenic, anti-parasitic, antiviral, insecticidal, and antioxidant properties [
16,
17,
19,
58‐
60]. Blum et al. [
19] evaluated the anti-
Helicobacter pylori effects of the NeO and suggested that this oil has promising bactericidal effects and would be considered for further treatment strategies against this bacterium. However, there is a little data about the effects of the Neem extract on
T. gondii. In this regard, Melo et al. [
23] evaluated the effects of the aqueous extract of Neem on intracellular development of
T. gondii in Vero cell, and observed a dramatically decrease in the number of the parasite in the parasitophorous vacuole without significant changes in the host cells. Nevertheless, due to some impediments, the wide biological usage of the Neem extracts has been limited. Actually, unspecific standard dosage, low stability, unknown side effects, low bioavailability, and low available scientific evidence seem to be the main concerns for the commercial development of the herbal medicine [
61]. For example, Mohammad Rahimi et al. [
36] showed that although the aqueous extract of
Mentha pulegium L. and
Rubus idaeus L. presented promising anti-
Toxoplasma effects, cell toxicity of the extracts, particularly,
R. idaeus L. limited employing of
R. idaeus L. for herbal therapy. The nanoformulation of the herbal extracts can not only increase the bioavailability, effectiveness, and target delivery, but also decrease the aforementioned limitations of the herbal extracts [
9,
62]. In the current study, we employed the SLNs to capsulate the NeO and increase its target delivery and efficiency. The SLNs are a group of nano-particles that are used to control the release and target delivery of drugs [
63]. In addition, this type of encapsulation increases the efficiency of natural products [
63,
64]. The SLNs, as a carrier, are reported to effectively increase the bioavailability of commercial drugs. Ud Din et al., [
29] demonstrated that SLNs formulation of ezetimibe increased the bioavailability of this drug compared to marketed product, while its stability remained without significant changes for three months.
The previous study in our laboratory suggested the promising effects of SLN capsulation. In this regard, Khosravi et al. [
33] suggested that the SLN capsulation of paromomycin decreased the cell toxicity and increased anti-
Toxoplasma activity of the drug. Similar to our protocol, Vijayan et al., [
28] synthesized NeO-SLNs with a high entrapment efficacy (82.1%) and investigated the anti-Acne microbes activity of NeO-SLNs that due to the promising results, they continued prolonged treatment of Acne. In addition, the results of a study by Kim et al., [
65] who formulated SLNs to carry
Houttuynia cordata for oral delivery, showed a low cell toxicity of SLNs loaded
H. cordata on caco-2 cell line, while its cumulative release continued to 50 h. The more effective delivery of SLNs loaded herbal extracts was also suggested by Vijayanand et al. [
66], who proposed encapsulation of
Hibiscus rosa sinensis extract by SLNs improve efficacy of the extract compared to crude extract, even at low dosage and in vivo model. In the line of these studies, our findings showed suitable anti-
T. gondii activity besides low cell toxicity in 100 μg/mL concentration of the NeO-SLNs. In addition, the NeO-SLNs promisingly limited the intracellular development of
Toxoplasma in Vero cells in 100 μg/mL concentration. Moreover, high entrapment and satisficing cumulative release of NeO-SLNs in our study suggested that encapsulation of the NeO by SLNs could be effective as an alternative therapy for not only
T. gondii tachyzoites, which are released in acute phase of toxoplasmosis, but also for cysts, which are formed in chronic stage. However, further studies are needed to evaluate the effects of NeO-SLNs in animal models and non-lethal (cyst-forming) strains.