Phytochemicals and potential therapeutic targets on toxoplasma gondii parasite

Identification of drug target in protozoan T. gondii is an important step in the development of chemotherapeutic agents. Likewise, exploring phytochemical compounds effective against the parasite can lead to the development of new drug agent that can be useful for prophylaxis and treatment of toxoplasmosis. In this review, we searched for the relevant literature on the herbs that were tested against T. gondii either in vitro or in vivo, as well as different phytochemicals and their potential activities on T. gondii. Potential activities of major phytochemicals, such as alkaloid, flavonoid, terpenoids and tannins on various target sites on T. gondii as well as other related parasites was discussed. It is believed that the phytochemicals from natural sources are potential drug candidates for the treatment of toxoplasmosis with little or no toxicity to humans

Deciphering the pharmacological effects of andrographolide on erythrocytes and Plasmodium falciparum 3D7 via metabolic changes by the ¹H NMR-based metabolomics approach

Malaria is a serious health problem associated with high morbidity and mortality rates, affecting millions of people across the world. The evolution of drug resistance among various strains of Plasmodium falciparum has thwarted the control efforts, thereby prompting scientists to seek for new and effective alternative therapeutic agents in order to forestall the menace caused by the parasite. This study was undertaken to evaluate and elucidate the pharmacological effects of andrographolide (AG) on P. falciparum 3D7 and erythrocytes using 1H-NMR-based metabolomics approach. The first part of the study was aimed at investigating the anti-plasmodium effect of AG against P. falciparum 3D7, its time-dependent effect as well as its impact on the cellular morphology of various stages of plasmodium intra-erythrocytic cycle as compared to the conventional drug chloroquine (CQ). The malaria drug sensitivity assay was carried out using pLDH and Giemsa-stained thin blood smears to determine the differences and the morphological changes at different time intervals during the growth stages of the parasite. In the second part of this study, the IC50 and time-dependent of AG and CQ were used to determine the pharmacological effects of AG and CQ on the metabolic change of uninfected erythrocytes (uRBCs), infected erythrocytes (iRBCs) and the P. falciparum 3D7 parasite in vitro. The 1H NMR-based metabolomics approach using Principal Component Analysis (PCA) and Orthogonal Partial Least Square discriminant analysis (OPLS-DA) were used. Overall, the results reveal that AG showed a good growth inhibitory effect (IC50 =4.14μM) that was substantially lower than that of CQ (IC50 =20.19 nM). Unlike CQ, which showed its utmost activity within the first 12 hours of the cycle, the AG effect was more prominent during the second 12 hours interval of the cycle (early trophozoites stage). Although AG failed to produce any effect on the morphology of the ring stage, it produced a noticeable change in the morphological appearance and the sizes of the mature trophozoites after 12 hours. In contrast, the rings and trophozoites stage of the parasites were fairly affected in the chloroquinetreated flasks within the first 12 hours and 24 hours of the cycle, respectively. Based on unsupervised data analysis PCA, the effects of AG and CQ on the metabolic changes of uRBCs showed a clear separation between all uRBCs samples with a total variance of 89.10%. A total of 28 and 32 metabolites were identified as biomarkers in uRBCs-AG and uRBCs-CQ, respectively. In uRBCs-AG, ten metabolic pathways were determined as disturbed metabolic pathways, including riboflavin metabolic pathway, D-Glutamine and D-glutamate metabolism, phenylalanine metabolism, arginine and proline metabolism, glutathione metabolism, arginine biosynthesis, citrate cycle, pyruvate metabolism, alanine, aspartate and glutamate metabolism and glycolysis/gluconeogenesis. In contrast, in uRBCs-CQ, nine metabolic pathways have been determined as disturbed metabolic pathways similar to uRBCs-AG except for glutathione metabolism. These findings suggest an evident relationship between AG and CQ associated with metabolic perturbations in uRBCs. The effects of AG and CQ on the metabolic changes of iRBCs, the PCA and OPLS-DA showed ideal differentiation between iRBCs samples treated and untreated. Thirty-five blood metabolites were identified from the 1H-NMR spectra analysis of iRBCs samples. The outcome of PCA showed clear discrimination between AG and CQ. Both PC1 and PC2 show a total variance of 77.10%. A total of 23 and 24 metabolites were identified as biomarkers in iRBCs-AG and iRBCs-CQ, respectively. The metabolic pathways analysis revealed ten metabolic pathways were identified as disturbed in all groups. The iRBCs untreated group had a high number of disturbed metabolic pathways, including alanine, aspartate and glutamate metabolism, glutathione metabolism, arginine and proline metabolism, and riboflavin metabolism. In the group of iRBCs-CQ, the disturbed metabolic pathways identified as alanine, aspartate and glutamate metabolism, arginine and proline metabolism, and glutathione metabolism. Whereas in the iRBCs-AG, the disturbed metabolic pathways identified include glyoxylate and dicarboxylate metabolism, glycine, serine and threonine metabolism, and histidine metabolism. The effects of AG and CQ on the metabolic changes of P. falciparum 3D7 in-vitro were identified. The results of multivariate data analysis show a clear discriminant between P. falciparum 3D7 samples treated and untreated. The model showed a total variance of 89.9% described by the PC1 and PC2. A total of 19 and 21 metabolites were identified as biomarkers in groups of P. falciparum 3D7-AG and P. falciparum 3D7- CQ, respectively. In P. falciparum 3D7-AG, very few metabolites biomarkers were observed, including threonine, ornithine, riboflavin, lactate and glutathione, compared to the group treated with CQ, which showed a high number of biomarkers. Analysis of the metabolic pathways reveals two metabolic pathways were significantly disturbed in P. falciparum 3D7-AG group; arginine and proline metabolism, and glutathione metabolism. In P. falciparum 3D7-CQ group, six disturbed metabolic pathways were identified: glyoxylate and dicarboxylate metabolism, glutathione metabolism, alanine, aspartate and glutamate metabolism, arginine biosynthesis, purine metabolism and citrate cycle. In conclusion, the present study is the first to report on the antimalarial activity of AG utilizing the 1H NMR-based metabolomics approach. Results from this study suggest that the disturbed metabolic pathways identified could well serve as drug targets for future development of andrographolide-based therapeutic agents against P. falciparum 3D7

Investigation of Andrographolide Effect on Non-Infected Red Blood Cells Using the 1H-NMR-Based Metabolomics Approach

Andrographolide (AG) has been shown to have several medicinal and pharmaceutical effects, such as antimicrobial, anti-inflammatory, antioxidant, anti-diabetic, and anti-malarial activities. Moreover, studies to assess the pharmacological effect of AG on the metabolic changes of uninfected red blood cells (uRBCs) have not yet been investigated. This study aims to evaluate the pharmacological effects of AG compared to chloroquine (CQ) on the metabolic variations of uRBCs in vitro using a proton nuclear magnetic resonance (1H-NMR)-based metabolomics approach coupled with multivariate data analysis (MVDA). Forty-one metabolites were successfully identified by 1H-NMR. The results of the unsupervised data analysis principal component analysis (PCA) showed ideal differentiation between AG and CQ. PC1 and PC2 accounted for 71.4% and 17.7% of the explained variation, respectively, with a total variance of 89.10%. Based on S-plot and VIP values, a total of 28 and 32 metabolites were identified as biomarkers in uRBCs-AG and uRBCs-CQ, respectively. In uRBCs treated with AG, ten metabolic pathways were determined to be disturbed, including riboflavin metabolism, d-glutamate and d-glutamine metabolism, phenylalanine metabolism, glutathione metabolism, proline and arginine metabolism, arginine biosynthesis, citrate cycle, glycolysis/gluconeogenesis, and pyruvate metabolism as well as alanine, aspartate, and glutamate metabolism. In contrast, in CQ-treated uRBCs, nine affected metabolic pathways were determined, which involved the same metabolic pathways for uRBCs-AG, except for glutathione metabolism. These findings suggest an evident relationship between AG and CQ associated with metabolic changes in intact RBCs after being exposed to the treatment. The metabolomics results could allow useful comprehensive insights into the underlying mechanism of the action of AG and CQ on red blood cells. Consequently, the 1H-NMR-based metabolomics approach was successfully utilized to identify the pharmacological effects of AG and CQ on the metabolic variations of uRBCs

Molecular detection and genetic diversity of toxoplasma gondii oocysts in cat faeces from Klang Valley, Malaysia, using B1 and REP genes in 2018

The major route for Toxoplasma gondii (T. gondii) infection is through the ingestion of foods contaminated with oocyst from cat faeces. The microscopic detection of T. gondii oocysts in cat faeces is challenging, which contributes to the failure of detecting or differentiating it from other related coccidian parasites. This study aims to detect T. gondii oocysts in cat faeces using two multicopy-target PCR assays and to evaluate their genetic diversity. Cat faecal (200) samples were collected from pet cats (PCs; 100) and free-roaming cats (FRCs; 100) within Klang Valley, Malaysia, and screened for coccidian oocysts by microscopy using Sheather’s sucrose floatation. PCR assays were performed on each faecal sample, targeting a B1 gene and a repetitive element (REP) gene to confirm T. gondii oocysts. Additionally, the PCR amplicons from the REP gene were sequenced to further confirm T. gondii-positive samples for phylogenetic analysis. Microscopy detected 7/200 (3.5%) T. gondii-like oocysts, while both the B1 gene and the REP gene detected 17/200 (8.5%) samples positive for T. gondii. All samples that were microscopically positive for T. gondii-like oocysts were also shown to be positive by both B1 and REP genes. The BLAST results sequenced for 16/200 (8.0%) PCR-positive T. gondii samples revealed homology and genetic heterogeneity with T. gondii strains in the GenBank, except for only one positive sample that did not show a result. There was almost perfect agreement (k = 0.145) between the two PCR assays targeting the B1 gene and the REP gene. This is the first report on microscopic, molecular detection and genetic diversity of T. gondii from cat faecal samples in Malaysia. In addition, the sensitivities of either the B1 gene or REP gene multicopy-target PCR assays are suitable for the accurate detection of T. gondii from cat faeces

A Review on the Prevalence of Toxoplasma gondii in Humans and Animals Reported in Malaysia from 2008–2018

Toxoplasmosis is a disease caused by the protozoan parasite Toxoplasma gondii (T. gondii). Human toxoplasmosis seroprevalence in Malaysia has increased since it was first reported in 1973 as shown in previous reviews of 1991 and 2007. However, over a decade since the last review, comprehensive data on toxoplasmosis in Malaysia is lacking. This work aimed at reviewing articles on toxoplasmosis research in Malaysia in order to identify the research gaps, create public awareness, and efforts made so far and proffer management options on the disease. The present review examines the available published research articles from 2008 to 2018 related to toxoplasmosis research conducted in Malaysia. The articles reviewed were retrieved from nine credible databases such as Web of Science, Google Scholar, ScienceDirect, PubMed, Scopus, Springer, Wiley online library, Ovid, and Cochrane using the keywords; Malaysia, toxoplasmosis, Toxoplasma gondii, toxoplasma encephalitis, seroprevalence, human immunodeficiency virus (HIV) patients, pregnant women, genotype strain, anti-toxoplasma antibodies, felines, and vaccine. The data highlighted seropositive cases from healthy community members in Pangkor Island (59.7%) and among migrant workers (57.4%) at alarming rates, as well as 42.5% in pregnant women. Data on animal seroprevalence were limited and there was no information on cats as the definitive host. Genetic characterization of Toxoplasma gondii from HIV patients; pregnant women, and domestic cats is lacking. This present review on toxoplasmosis is beneficial to researchers, health workers, animal health professionals, and policymakers. Therefore, attention is required to educate and enlighten health workers and the general public about the risk factors associated with T. gondii infection in Malaysia