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International Journal for Parasitology: Drugs and Drug Resistance logoLink to International Journal for Parasitology: Drugs and Drug Resistance
. 2024 Aug 13;26:100561. doi: 10.1016/j.ijpddr.2024.100561

Inhibition of Giardia duodenalis by isocryptolepine -triazole adducts and derivatives

Supaluk Popruk a, Jumreang Tummatorn b,c, Suthasinee Sreesai d, Sumate Ampawong e, Tipparat Thiangtrongjit f, Phornpimon Tipthara g, Joel Tarning g,h, Charnsak Thongsornkleeb b,c, Somsak Ruchirawat b,c, Onrapak Reamtong f,⁎
PMCID: PMC11377146  PMID: 39151240

Abstract

Giardia duodenalis, a widespread parasitic flagellate protozoan causing giardiasis, affects millions annually, particularly impacting children and travellers. With no effective vaccine available, treatment primarily relies on the oral administration of drugs targeting trophozoites in the small intestine. However, existing medications pose challenges due to side effects and drug resistance, necessitating the exploration of novel therapeutic options. Isocryptolepine, derived from Cryptolepis sanguinolenta, has demonstrated promising antimicrobial and anticancer properties. This study evaluated eighteen isocryptolepine-triazole adducts for their antigiardial activities and cytotoxicity, with ISO2 demonstrating potent antigiardial activity and minimal cytotoxicity on human intestinal cells. Metabolomics analysis revealed significant alterations in G. duodenalis metabolism upon ISO2 treatment, particularly affecting phospholipid metabolism. Notably, the upregulation of phytosphingosine and triglycerides, and downregulation of certain fatty acids, suggest a profound impact on membrane composition and integrity, potentially contributing to the parasite's demise. Pathway analysis highlighted glycerophospholipid metabolism, cytochrome b5 family heme/steroid binding domain, and P-type ATPase mechanisms as critical pathways affected by ISO2 treatment, underscoring its importance as a potential target for antigiardial therapy. These findings shed light on the mode of action of ISO2 against G. duodenalis and provide valuable insights for further drug development. Moreover, the study also offers a promising avenue for the exploration of isocryptolepine derivatives as novel therapeutic agents for giardiasis, addressing the urgent need for more effective and safer treatment options.

Keywords: Giardia duodenalis, Isocryptolepine -triazole adducts, Metabolomics

Graphical abstract

Image 1

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1. Introduction

Giardia duodenalis (syn. G. lamblia, G. intestinalis), a parasitic flagellate protozoan, infects many mammalian hosts and causes giardiasis with a worldwide distribution (Fava et al., 2016), (Hajare et al., 2022), (Krumrie et al., 2022), (Rodríguez-Morales et al., 2016). Nearly 300 million human cases of diarrhea with G. duodenalis are reported annually (Einarsson et al., 2016),(Fink et al., 2020). Children and travellers are the risk groups for giardia infection (Júlio et al., 2012). The prevalence of giardiasis ranges from 2 to 7% in developed countries and 20–30% in developing countries (Leung et al., 2019). There are 2 morphological forms of G. duodenalis in its life cycle: cyst and trophozoite (Benchimol et al., 2022). Cyst form is an infective stage responsible for the transmission of giardiasis. Transmission is mainly ingestion via contaminated water and food with infectious cysts or by direct fecal-oral contact (Leung et al., 2019). Excystation occurs in the small intestine, where the cysts transform into trophozoites. To date, no anti-Giardia vaccine exists for human use. Treatment of giardiasis in humans is oral drug administration for eliminating the trophozoites that attach to the gastro-epithelial cells of the proximal small intestine (Ankarklev et al., 2010),(Riches et al., 2020). First-line treatment of giardiasis is nitroimidazole derivatives (metronidazole, tinidazole, secnidazole and ornidazole), benzimidazoles, nitazoxanide, furazolidone, quinacrine, chloroquine and paromomycin (Bahadur et al., 2014),(Mørch and Hanevik, 2020). Although these drugs are available in use, many problems are reported regarding cost-effectiveness and undesirable side effects such as metallic taste, headache and dry mouth, and to a lesser extent, nausea, glossitis, urticarial, pruritus and dark colored urine, and carcinogenic, teratogenic, and embryonic properties (Leung et al., 2019), (Mørch and Hanevik, 2020), (Pintong et al., 2020). In addition, a high rate of ineffective giardiasis treatment due to drug resistance has been reported (Mørch and Hanevik, 2020),(Lalle and Hanevik, 2018). Hence, the discovery of potential new pharmaceutical products is required for giardia treatment.

Cryptolepis sanguinolenta plant has been traditionally used for malaria treatment in the West and Central Africa. Isocryptolepine, a naturally occurring alkaloid, was extracted from C. sanguinolenta root (Pousset et al., 1995). The IC50 of isocryptolepine against 3D7 parasite line was 665 nM (Whittell et al., 2011). Not only antimalarial activity, isocryptolepine analogues also showed anticancer (Aksenov et al., 2018) and antifungal properties (Li et al., 2019). According to mode of action of tsocryptolepine derivatives, they have been reported to target human topoisomerase II in various cancer cells (Riou et al., 1991). Proteomics analysis has shown that treatment with an isocryptolepine derivative in P. falciparum results in significant changes in pathways related to ribosomes, proteasomes, metabolic pathways, amino acid biosynthesis, oxidative phosphorylation, and carbon metabolism. Furthermore, transmission electron microscopy revealed a clear loss of ribosomes in isocryptolepine derivative -treated P. falciparum (Rujimongkon et al., 2019). Additionally, the linear indolequinoline alkaloids cryptolepine, neocryptolepine, and isocryptolepine can act as DNA-intercalating agents, inhibiting DNA replication and transcription (Kraus and Guo, 2010). To improve biological activity of isocryptolepine derivatives, it is well established that 1,2,3-triazole is a crucial linker in novel drug discovery (Kharb et al., 2011), as it remains stable under both basic and acidic conditions. In some instances, triazole also enhances biological activities by binding with biological targets through hydrogen bonding, noncovalent, or van der Waals interactions (Kerru et al., 2020). Recently, Aroonkit P and co-workers synthesized isocryptolepine derivatives with triazole and demonstrated their activities against chloroquine- and mefloquine-resistant Plasmodium falciparum strains. Moreover, these derivatives also exhibited anticancer activity against HepG2, HuCCA-1, MOLT-3, and A549 cancer cell lines (Aroonkit et al., 2015). In addition, Rodphon W developed isocryptolepine-triazole derivatives that exhibit strong inhibition against cancer cell lines, including HuCCA-1, HepG2, and A549 (Rodphon et al., 2021). Accordingly, there is interest in assessing additional antipathogenic activities of isocryptolepine derivatives. In this study, eighteen isocryptolepine-triazole adducts from Rodphon W study were evaluated for their antigiardial activities and cytotoxicity. In addition, the mode of action of isocryptolepine derivative was assessed by metabolomics and transmission electron microscopy.

2. Material and methods

2.1. G. duodenalis culture

G. duodenalis trophozoites contained in cryotube were thawed at 37 °C in a water bath for 1 min. Then, we transferred thawed G. duodenalis trophozoites into the 15 mL sterile conical centrifuge tube containing 12 ml of modified TYI-S-33 medium (Trypticase-yeast extract-iron-serum medium) (Keister, 1983) and mixed gently by inversion. After that, we centrifuged the tube at 1000×g for 10 min at 4 °C. The pellet was used and transferred to 15 mL of a glass tube screw cap containing warm modified TYI-S-33 medium sealing with paraffin and incubated at 37 °C. The trophozoites were examined for cell growth and cell viability after incubation for 24 h by using inverse microscopy. Log-phase cultures were harvested by placing on ice for 20 min and centrifuging at 1500 rpm, at 4 °C for 15 min. The trophozoites were counted in a hemocytometer and used for subsequent experiments.

2.2. In vitro antigiardial assay

Each compound and metronidazole (Sigma-Aldrich, St Louis, MO: positive control) were dissolved in 100% dimethyl sulfoxide (DMSO), and two-fold serial dilutions were made. Culture medium alone was used as a blank and 0.25% DMSO was used as negative control which this concentration did not affect trophozoites. Briefly, various concentrations of each compound, metronidazole (positive control), negative control, and blank were added to white opaque-walled 96-well microplates (PerkinElmer, U.S.A.). Then, 2 × 105 trophozoites were added to each well, except blank wells, to give a final volume of 100 μL. The final concentration of the DMSO was 0.25% and this did not affect the test. All experiments were performed in triplicates. The plates were sealed and incubated at 37 °C for 48 h under anaerobic conditions in 2.5-L Pack-Rectangular Jars (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan). The tested microplates were incubated for 48 h and subsequent 100 μL BacTiter-GloTM Microbial Cell Viability Assay fluid was added directly to each well. The tested microplates were mixed on an orbital shaker and incubated for 20 min at 37 °C before trophozoite viability was recorded using luminescence.

The percentage trophozoite viability at various concentrations of each compound and metronidazole was determined using the following formula.

  • (i)

    %cell survival = [(sample luminescence − culture medium luminescence)/(non-treated control luminescence − culture medium luminescence)] × 100

  • (ii)

    %inhibition = 100 – %trophozoites that survived

2.3. Cytotoxicity

Immortalized cell line of human colorectal adenocarcinoma cells (Caco-2) was cultured in a 96-well plate in Eagle's minimum essential medium (EMEM) with 10% fetal bovine serum (HyClone, GE Healthcare Life Science, USA) at 37 °C with 5% CO2 for 24 h. The 0.5 % DMSO, isocryptolepine-triazole derivatives ranging from 10 ng/ml to 100 μg/ml were prepared in EMEM, added to the cells, and incubated for 24 h. Each well was filled with 10 μl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (AppliChem GmbH, Germany), which was then incubated for 4 h at 37 °C with 5% CO2. To dissolve the formazan crystal, all of the solution was replaced with a solvent (4 mM HCl, 0.1% Nonidet P-40) in isopropanol, and the measurement was made at 590 nm using 620 nm as a reference wavelength. The absorbance of the treated groups was compared to the untreated control group to determine the percentage of viable cells. The 50% cytotoxic concentration (CC50) was determined by GraphPad Prism 9.

2.4. Metabolomics analysis

G. duodenalis treated with 0.25% DMSO and EC50 of ISO2 compound were homogenized in 500 μL methanol. The lysates were centrifuged at 800 g for 1 min at 4 °C after being snap-frozen in liquid nitrogen and thawed. The same process was used to extract the pellet once more. The supernatant from the second extraction was combined with the one from the first. The pellet was dissolved in 250 μL of deionized water and frozen-thawed by liquid nitrogen. The supernatant was obtained by centrifugation at 15,000 g for 1 min at 4 °C was added to the previous extraction. To get rid of the debris, the pooled supernatants were centrifuged at 15,000 g for 1 min at 4 °C. The clear supernatant was collected and dried in a speed vacuum (Tomy Digital Biology, Tokyo, Japan). The metabolite pellet was resuspended in 200 μL of mobile phase A: B at a ratio of 50:50 (vol/vol). The samples were subjected to ultra-high performance liquid chromatography (UHPLC; Agilent 1260 Quaternary pump, Agilent 1260 High-Performance Autosampler, and Agilent 1290 Thermostatted Column Compartment SL, Agilent Technologies) connected with a quadrupole time-of-flight mass spectrometer (Q-TOF-MS) (TripleTOF 5600+, SCIEX, US). The mobile phase A and B was 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. The pre-column and analytical column were an ACQUITY UPLC HSST3, 2.1 × 5 mm, 1.8 μM column (Waters) and C18 reversed-phase column (ACQUITY UPLC HSST3, 2.1 × 100 mm, 1.8 μM, Waters), respectively. The gradient was started at 5%–100% mobile phase B in 20 min. The UHPLC-Q-TOF-MS system was controlled by Analyst Software version 1.7 (SCIEX). The acquisition was done in positive (+ESI) and negative (-ESI) electrospray ionization modes. The quality control (QC) samples were prepared by pooling equal aliquots of each metabolite sample and injected before, during, and after sample analysis. The.wiff and.wiff.scan files were analyzed by the XCMS online software version 3.7.1 (The Scripps Research Institute, CA, USA) and MetaboAnalyst online software version 5.0 (https://www.metaboanalyst.ca/). Metabolite quantification was filtered using the "Interquantile range (IQR)". Quantile normalization, cube root data transformation, and data range scaling were then used to normalize the data. Principal Component Analysis (PCA), Partial Least Squares-Discriminant Analysis (PLS-DA), and Volcano Plot were performed for data visualization. The Volcano plot was created using the log2 of the fold change and the -log of the p-value. Differential metabolites were distinguished using the p-value<0.01 and the specific threshold of ≥ 2fold change. The STITCH database version 5.0 (http://stitch.embl.de/) was carried out for pathway analysis of differential metabolites with a statistical significance threshold of p-value less than 0.01.

2.5. Fluorescent microscopy

Ethidium bromide/Acridine orange (EB/AO) staining was used to identify the ISO2 compound-induced apoptotic cells. The 50 μL of giardia suspension were combined with 2 μL of the 100 μg/mL EB and AO dye mixture. Under a fluorescence microscope, cellular morphology and the number of apoptosis were assessed in at least ten high-power fields (400X)/group settings. The distinct stained colors including orange for apoptotic cells, red for necrotic cells, and green for normal cells represented distinct stages of the cell architecture.

2.6. Conventional transmission electron microscopy

In each group, the giardia pellets were fixed for 1 h at room temperature using 2.5% glutaraldehyde and 1% osmium tetroxide, respectively. The fixed pellets were dehydrated using ethanol, infiltrated, embedded in LR white resin (EMS, USA), polymerized for 24–48 h at 65 °C in an oven, and then sliced into thicknesses of 100 nm. Using a transmission electron microscope (Hitachi; model HT7700, Japan), the sections were inspected to find alterations in the parasitic ultrastructure that occurred during the treatment process.

3. Results and discussion

3.1. In vitro antigiardial Activity and cytotoxicity of isocryptolepine-triazole adducts

The structures of eighteen isocryptolepine-triazole adducts were demonstrated in Fig. 1 (Rodphon et al., 2021). All derivatives were assayed for their in vitro antigiardial activity. In addition, they were assessed cytotoxicity on Caco-2 cells. The results are demonstrated in Table 1.

Fig. 1.

Fig. 1

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The structures of isocryptolepine-triazole adducts.

Table 1.

Half-maximal inhibitory concentration (IC50) of isocryptolepine-triazole adducts on G. duodenalis trophozoite stage, 50% cytotoxic concentration (CC50) on Caco-2 human intestinal cell and selectivity index (SI) of isocryptolepine-triazole adducts.

Compound IC50
 CC50
 SI (CC50/IC50)
μg/mL μM μg/mL μM
ISO1 0.7 ± 0.0 1.6 ± 0.0 1.7 ± 0.3 4.2 ± 0.6 2.5
ISO2 0.1 ± 0.0 0.2 ± 0.0 4.1 ± 0.1 8.7 ± 0.3 37.2
ISO3 0.6 ± 0.0 1.5 ± 0.0 7.2 ± 0.6 16.0 ± 1.4 11.0
ISO4 0.6 ± 0.0 1.4 ± 0.0 7.2 ± 0.1 16.9 ± 0.2 11.7
ISO5 1.2 ± 0.0 2.7 ± 0.0 7.2 ± 0.1 16.4 ± 0.3 6.0
ISO6 1.5 ± 0.0 3.4 ± 0.0 7.3 ± 0.2 16.4 ± 0.5 4.8
ISO7 1.4 ± 0.0 3.0 ± 0.1 7.4 ± 0.5 16.0 ± 1.1 5.3
ISO8 1.0 ± 0.0 2.1 ± 0.0 7.3 ± 0.0 15.9 ± 0.1 7.4
ISO9 >2.5 >5.0 7.1 ± 0.2 14.7 ± 0.5 –
ISO10 1.3 ± 0.0 2.7 ± 0.1 8.4 ± 0.3 18.0 ± 0.6 6.7
ISO11 1.0 ± 0.0 2.19 ± 0.02 7.6 ± 0.8 17.4 ± 1.7 8.0
ISO12 2.5 ± 0.3 5.6 ± 0.7 7.6 ± 0.1 17.1 ± 0.2 3.1
ISO13 >2.5 >5.0 10.0 ± 0.2 20.4 ± 0.3 –
ISO14 >2.5 >5.0 5.7 ± 0.3 11.3 ± 0.7 –
ISO15 >2.5 >5.0 9.2 ± 0.1 20.8 ± 0.2 –
ISO16 1.0 ± 0.0 2.3 ± 0.0 22.7 ± 1.6 49.5 ± 3.4 21.6
ISO17 1.2 ± 0.0 2.3 ± 0.0 2.4 ± 0.1 5.2 ± 0.2 2.0
ISO18 1.5 ± 0.0 3.3 ± 0.1 1.7 ± 0.2 3.8 ± 0.4 6.7
Metronidazole 2.0 ± 0.3 11.7 ± 2.0 >100.0 >100.0 –
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Metronidazole was employed as the positive control in this experiment, presenting an IC50 of 2.01 μg/mL, consistent with findings from other studies (Popruk et al., 2023),(Campanati and Monteiro-Leal, 2002). All isocryptolepine-triazole adducts were categorized as having highly active antigiardial activity. Among these compounds, ISO2 demonstrated the most antigiardial activity with the half-maximal inhibitory concentration (IC50) against the G. duodenalis trophozoite stage as shown in Table 1. ISO1, ISO2, and ISO3 each contain phenyl, 2-iodophenyl, and 4-methoxyphenyl groups, respectively, with the iodophenyl group demonstrating significantly greater activity than the others. This result presents an opportunity for further investigation, such as exploring the effects of different substitution patterns, including 2-substituted phenyl derivatives (e.g., Me, Cl, F). In addition, the antigiardial activity of isocryptolepine itself should be explored, as it serves as the core structure before derivatization. Further investigations should be conducted to measure and compare the antigiardial activities of isocryptolepine with its fluoro- and bromo-substituted derivatives. In terms of the 50% cytotoxic concentration (CC50) on Caco-2 human intestinal cell, ISO16 exhibited the highest CC50, suggesting lower cytotoxicity. Calculating the selectivity index (SI), ISO2 yielded a value of 37.21. The isocryptolepine-triazole adducts showed moderate antigiardial activity except for ISO2 which was significantly more active. Due to ISO2 selectivity between humans and G. duodenalis, it was chosen for further investigation into its mode of action using metabolomics and transmission electron microscopy.

3.2. Metabolomics analysis

The mechanisms of ISO2 on G. duodenalis were explored by metabolomics analysis. The metabolite profiles of 0.25% DMSO (control) and ISO2 at IC50 treated G. duodenalis trophozoites were analyzed. After data processing and analysis, the results were visualized in principal component analysis (PCA) and partial least squares–discriminant analysis (PLS-DA) as shown in Fig. 2.

Fig. 2.

Fig. 2

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Pairwise analysis of metabolomic data using principal component analysis (A) and partial least squares–discriminant analysis (B). Red and green represent the data from the control and ISO2 treatment, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

The metabolomics profiles of control and ISO2 treated G. duodenalis differed as a result of the PCA and PLS-DA analysis. A total of 13,965 features were identified from mass spectrometric data. Among them, 360 and 448 were up- and down-regulated features, respectively (Supplemental Table 1). A volcano plot was created to depict the differential features chosen for p-value 0.01 and folded change 2 after exposure to ISO2 (Fig. 3). By using the MATLIN database, 79 and 179 were identified as up- and down-metabolites, respectively (Supplementary Table 1).

Fig. 3.

Fig. 3

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Volcano plots demonstrating differential metabolites of G. duodenalis after ISO2 treatment. Horizontal lines represent p-values equal to 0.01. Vertical lines represent fold changes equal to 2 and −2, respectively.

According to the degree of change (two-fold change) vs. statistical significance (0.01 p-value). The top twenty up- and down-regulated metabolites of G. duodenalis after treatment with ISO2 are presented in Table 2, Table 3, respectively.

Table 2.

Top-twenty up-regulated metabolites of G. duodenalis after treatment with ISO2.

No. Potential metabolites m/z Retention time (min) Mass error (ppm) Fold change p-value
1 Phytosphingosine 318.3 6.6 1 10.03 0.0020
2 TG(20:4/20:5/20:5)[iso3] 991.674 8.48 2 9.40 0.0000
3 mycophenolic acid (C24) 402.3937 13.73 1 8.79 0.0014
4 2-amino-14,16-dimethyloctadecan-3-ol 314.3415 11.7 1 7.98 0.0007
5 C16 Sphinganine 274.2743 6.66 1 6.83 0.0098
6 4-oxo-docosanoic acid 372.3474 14.84 1 6.39 0.0081
7 2-amino-14,16-dimethyloctadecan-3-ol 314.342 13.92 1 5.94 0.0018
8 3-Methylbutyl pentadecanoate 311.296 15.7 2 5.77 0.0000
9 6,10,13-Trimethyltetradecyl 3-methylbutanoate 358.3682 13.7 1 5.55 0.0022
10 PA(14:1(9Z)/18:4(6Z,9Z,12Z,15Z)) 637.3905 10.19 5 4.29 0.0016
11 PG(18:1(9Z)/0:0) 509.2895 8.74 2 4.04 0.0000
12 PC(15:0/0:0) 480.3098 8.44 0 3.90 0.0000
13 PE(18:1(9Z)/0:0) 478.2941 8.67 0 3.90 0.0026
14 GlcCer(d18:2/20:0) 753.6126 8.47 1 3.68 0.0093
15 LysoPE(0:0/18:2(9Z,12Z)) 476.2786 7.75 1 3.60 0.0004
16 1-heptadecanoyl-sn-glycero-3-phosphocholine 508.3412 10.52 1 3.39 0.0048
17 PS(19:0/0:0) 538.3157 7.45 1 3.31 0.0015
18 LysoPE(0:0/18:0) 480.3102 10.45 1 3.22 0.0017
19 19-oxo-eicosanoic acid 344.316 11.66 0 3.17 0.0071
20 PC(15:0/0:0) 480.3096 9.15 0 3.09 0.0076
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Table 3.

Top-twenty down-regulated metabolites of G. duodenalis after treatment with ISO2.

No. Potential metabolites m/z Retention time (min) Mass error (ppm) Fold change p-value
1 9Z-Tritriacontene 485.504 19.29 4 −493.10 0.0013
2 PS(P-20:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 892.5419 12.2 2 −324.98 0.0000
3 1-Hentriacontene 457.4731 15.52 3 −297.44 0.0000
4 PI(O-18:0/0:0) 551.3365 12.2 2 −73.89 0.0000
5 PS(P-20:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 892.5422 12.5 2 −64.78 0.0000
6 PS(13:0/18:4(6Z,9Z,12Z,15Z)) 713.4256 12.2 2 −58.45 0.0000
7 MG(0:0/22:1(13Z)/0:0) 740.521 14.48 0 −49.16 0.0002
8 8E-Heneicosene 312.3626 15.01 0 −31.67 0.0010
9 Lignoceric acid 740.521 16.72 1 −28.82 0.0015
10 PI(13:0/12:0) 695.4153 12.2 2 −26.01 0.0000
11 Amino a: Val Leu Glu Ser Phe 657.3216 6.07 0 −22.78 0.0000
12 3-oxohexacosanoic acid 428.41 16.76 0 −22.47 0.0019
13 PS(15:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 740.521 12.2 1 −21.32 0.0015
14 1-Octadecanamine 740.521 13.86 0 −16.65 0.0006
15 PS(O-20:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) 894.5568 13.02 3 −14.38 0.0001
16 PI(P-16:0/20:5(5Z,8Z,11Z,14Z,17Z)) 885.4868 11.41 0 −14.34 0.0036
17 3-O-trans-Feruloyleuscaphic acid 740.521 12.2 1 −12.84 0.0003
18 methyl-10-hydroperoxy-8E,12Z,15Z-octadecatrienoate 307.2272 12.2 0 −12.13 0.0000
19 thevetin B 900.4577 11.41 1 −11.89 0.0068
20 13R-Methyl-6E-heneicosene 326.3787 12.27 2 −11.66 0.0006
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As measured by the fold change, the most up-regulated metabolites following treatment with ISO2 were phytosphingosine, TG(20:4/20:5/20:5)[iso3] and mycolipanolic acid (C24). In addition, several phospholipid molecules were also up-regulated after ISO2 exposure such as PA(14:1(9Z)/18:4(6Z,9Z,12Z,15Z)), PG(18:1(9Z)/0:0), PC(15:0/0:0) and PE(18:1(9Z)/0:0). Phytosphingosine serves as a sphingoid base, constituting a foundational component of complex sphingolipid structures. Its prevalence is notable in both plants and fungi, while also found in animals (Park et al., 2003). The evaluation of gene expression levels of sphingolipid biosynthesis in C. albicans after miconazole (a fungicidal imidazole) treatment was conducted, with a comparative analysis against untreated C. albicans. Notably, YDC1 associated with the synthesis of phytosphingosine demonstrated a 2.1-fold upregulation of cells after miconazole treatment. Moreover, the introduction of 10 nM phytosphingosine-1-phosphate led to a marked reduction in the intracellular miconazole concentration and substantially enhanced the miconazole resistance in C. albicans (Vandenbosch et al., 2012). In G. duodenalis, there was an upregulation of phytosphingosine after exposure to ISO2, similar to the upregulation observed in C. albicans during miconazole exposure. Furthermore, the involvement of phytosphingosine may contribute to a decrease in ISO2 cellular concentration, potentially reducing its toxicity, aligning with similar observations in C. albicans. TG(20:4/20:5/20:5)[iso3] is a triglyceride composed of glycerol and three fatty acids. Whereas mycolipanolic acid (C24) is a long-chain fatty acid. The growth of giardia is adversely affected by free fatty acids originating from phospholipids and triglycerides (Das et al., 1988). Research findings propose that lauric acid (C12:0), a medium-chain fatty acid, exhibits antigiardial properties even at relatively low concentrations (Rayan et al., 2005). In vitro experiments have revealed that normal human milk effectively eliminates G. duodenalis trophozoites, with the primary mechanism being the release of free fatty acids (Reiner et al., 1986). The up-regulation of free fatty acid (mycolipanolic acid) and triglyceride (TG(20:4/20:5/20:5)[iso3]), hydrolyzing to release free fatty acids, may be one of ISO2 antigiardial activity. Several phospholipid such as PA(14:1(9Z)/18:4(6Z,9Z,12Z,15Z)), PG(18:1(9Z)/0:0), PC(15:0/0:0) and PE(18:1(9Z)/0:0) were also up-regulated after ISO2 exposure. Phospholipids serve as integral components of cellular membranes. While numerous parasites acquire these phospholipids from their host, the majority also engage in de novo phospholipid synthesis or undergo substantial modifications to a significant portion of the scavenged phospholipids (Vial et al., 2003). A novel class of phospholipid mimetics, demonstrating inhibitory effects on the growth of P. falciparum, as well as Leishmania and Trypanosoma species. The inference of phospholipid metabolism could affect parasite survival (González-Bulnes et al., 2011). Ether phospholipid-dinitroaniline demonstrated a comprehensive antiparasitic profile by disrupting parasite membrane phospholipids (Magoulas et al., 2021). The differential expression of phospholipids could potentially impact the formation of the G. duodenalis membrane, ultimately resulting in the death of the parasite. The most down-regulated metabolites after treatment with ISO2 were 9Z-Tritriacontene, PS(P-20:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)) and 1-hentriacontene. Some organic acids were also down-regulated after ISO2 exposure such as 3-oxohexacosanoic acid and lignoceric acid. There is a scarcity of information regarding 9Z-Tritriacontene, PS(P-20:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), and 1-hentriacontene in parasites. Concerning 3-oxohexacosanoic acid, it is an elongated fatty acid. In the biosynthetic pathways of hexacosanoic acid in both Saccharomyces cerevisiae and Candida albicans, it is considered essential for yeast (Janik et al., 2021), (Kohlwein et al., 2001). Given the significance of hexacosanoic acid for organism viability, the down-regulation of 3-oxohexacosanoic acid may similarly impact the survival of G. duodenalis. Lignoceric acid belongs to the class of organic compounds known as very long-chain fatty acids. These are fatty acids with an aliphatic tail that contains at least 22 carbon atoms. Reducing the expression of fatty acid synthase especially, very-long-chain fatty acids enhances the resistance of S. cerevisiae cells to H2O2 (Matias et al., 2007). The down-regulation of lignoceric acid might serve as a mechanism for G. duodenalis to tolerate the toxicity of ISO2.

Using the MetaboAnalyst software, a comprehensive pathway analysis was conducted on all differentially expressed metabolites following exposure to ISO2. The outcomes underscored the significance of glycerophospholipid metabolism as a pivotal pathway, evident from a p-value below 0.05, as depicted in Fig. 4. Glycerophospholipids, the principal constituents of the parasite membrane, are primarily derived from the enzymatic machinery within the parasite. This process depends on the scavenging and subsequent metabolism of polar heads and fatty acids. The glycerophospholipid metabolism was considered as antiparasitic drug target on many parasites such as Brugia pahangi (Srivastava et al., 1987), Brugia patei (Srivastava et al., 1987), Plasmodium spp. (Déchamps et al., 2010), Toxoplasma gondii (He et al., 2024), Trichinella papuae (Mangmee et al., 2020) and Brugia malayi (Niyomploy et al., 2022). Additionally, the STITCH bioinformatics tool was employed for pathway analysis on diverse metabolites (Fig. 5), revealing cytochrome b5-like Heme/Steroid binding domain as an up-regulated pathway. While P-type ATPase was a down-regulated pathway after ISO2 exposure.

Fig. 4.

Fig. 4

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Pathway analysis using MetaboAnalyst software demonstrating differential metabolites of G. duodenalis after ISO2 treatment. Glycerophospholipid metabolism is significant pathway with a statistically significant p-value below 0.05.

Fig. 5.

Fig. 5

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Pathway analysis by Stitch software of up- (A) and down-regulated metabolites (B) in G. duodenalis after ISO2 treatment. The significant gene ontologies were enriched and presented in the red line. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

In Peronophythora litchi, a cytochrome b5 family heme/steroid binding domain-containing domain regulates mycelial growth, pathogenicity, and tolerance in G. duodenalis after ISO2 treatment (Li et al., 2021). In Toxoplasma gondii, the antiparasitic efficacy of dehydroepiandrosterone may arise from its interaction with the cytochrome b5 family heme/steroid binding domain-containing protein. (Muñiz-Hernández et al., 2021). The increase in expression of the cytochrome b5-like Heme/Steroid binding domain in G. duodenalis may potentially respond to oxidative stress after ISO2 treatment. P-type ATPases play a crucial role in maintaining and regulating cellular ion homeostasis and membrane lipid asymmetry by transporting ions and phospholipids against concentration gradients using ATP hydrolysis as an energy source. In the life cycles of human pathogenic trypanosomatids, which undergo notable and sudden shifts in their surrounding environments, P-type ATPases play a crucial role. These P-type ATPase subfamilies are correlated with trypanosomatid growth, adaptation, infectivity, and survival. (Meade, 2019). In Leishmania amazonensis, the inhibition of intracellular leishmanial growth by furosemide, achieved through the inhibition of parasite P-type ATPase and activation of macrophage ROS, may elucidate its singular therapeutic efficacy and its effectiveness in combination with SSG against murine cutaneous leishmaniasis (Arruda-Costa et al., 2017). Similar to Leishmania and Trypanosoma, the decrease in G. duodenalis P-type ATPase expression following ISO2 treatment could impact parasite growth and survival. Conducting an in-depth exploration of the pathways associated with ISO2. The findings highlighted glycerophospholipid metabolism, cytochrome b5 family heme/steroid binding domain and P-type ATPase as primary mechanisms of ISO2 influencing the altered metabolites. Additionally, the potential identification of glycerophospholipid metabolism, cytochrome b5 family heme/steroid binding domain and P-type ATPase might be the potential routes for novel drug discovery emerging from these results. However, isocryptolepine has been reported to inhibit the formation of beta-haematin and act as a DNA chelator (Nuthakki et al., 2022). One limitation of this study is that experiments were not conducted to confirm the activities of the isocryptolepine derivatives or their relevance to the observed activity against G. duodenalis.

3.3. Electron microscopy

EB/AO staining indicated the apoptotic-medicated effect of the extraction by the significant increase in the number of apoptotic cells and the significant decrease in the number of intact cells when compared with no treatment group (Fig. 6B–C). However, the necrotic cell number was identical between the two groups (Fig. 6D). Electron microscopic study revealed the ultrastructural alterations of giardia after exposure with extraction mainly represented by cytoplasmic and nucleolar damage and degeneration (Fig. 6E–H). Several studies reported that the ultrastructure of the parasite changed after antiparasitic treatment. On the malaria parasite, the ultrastructural alterations after chalcone derivatives treatment observed indicate substantial disruption of various parasite membranes, such as those surrounding the nucleus, mitochondria, and food vacuole (Li et al., 2021). In addition, the antiparasitic efficacy and resultant ultrastructural changes were also induced by organoruthenium complexes against Leishmania amazonensis (Sinha et al., 2020). Interestingly, the novel toxoplasmicidal properties of dehydroepiandrosterone on extracellular tachyzoites through meticulous ultrastructural examinations of treated parasites. A distinctive expression profile in the presence of dehydroepiandrosterone, notably manifesting in the upregulation of a progesterone receptor membrane component harboring a cytochrome b5 family heme/steroid binding domain-containing protein (Colina-Vegas et al., 2019). This discovery echoes the observed upregulation of a cytochrome b5 family heme/steroid binding domain-containing protein and the consequential ultrastructural changes observed in G. duodenalis following ISO2 treatment. The mechanisms underlying the actions of ISO2 and dehydroepiandrosterone (DHEA) may intersect, suggesting a potential convergence in their modes of action.

Fig. 6.

Fig. 6

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Apoptotic staining and giardia ultrastructural changes after extraction exposure compared with non-treatment: Difference patterns of cellular architecture were characterized by EB/AO staining (A); intact cell with whole green staining, cytoplasmic apoptosis with diffused orange staining in the cytoplasm, nucleolar apoptosis with orange nuclei, and necrosis with absolutely red staining. Ultrastructural changes of giardia exposure with the extraction were demonstrated by electron microscopic study (E–H); intact cell (E), nucleolar degeneration (F), both cytoplasmic and nucleolar degeneration (G), and cytoplasmic degeneration (H). Dash-line indicated a degenerative area, ****: p value < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

4. Conclusion

In conclusion, the study evaluated the antigiardial activity and cytotoxicity of eighteen isocryptolepine-triazole adducts, with ISO2 demonstrating potent antigiardial activity and promising selectivity. Metabolomics analysis revealed significant alterations in G. duodenalis metabolites following ISO2 treatment, particularly in pathways related to glycerophospholipid metabolism, cytochrome b5 family heme/steroid binding domain, and P-type ATPase. These findings provide valuable insights into the mode of action of ISO2 against G. duodenalis and highlight its potential as a promising antigiardial compound. Further research on ISO2 and its derivatives could lead to the development of novel and effective treatments for giardiasis.

Funding

This research project has been funded by Mahidol University (Fundamental Fund: fiscal year 2024 by the National Science Research and Innovation Fund (NSRF) to OR.

Data availability

Data can be provided upon request.

CRediT authorship contribution statement

Supaluk Popruk: Writing – review & editing, Writing – original draft, Resources, Methodology, Investigation, Conceptualization. Jumreang Tummatorn: Writing – review & editing, Writing – original draft, Resources, Investigation, Conceptualization. Suthasinee Sreesai: Software, Investigation. Sumate Ampawong: Validation, Methodology, Investigation. Tipparat Thiangtrongjit: Investigation. Phornpimon Tipthara: Software, Methodology, Investigation. Joel Tarning: Resources, Methodology, Conceptualization. Charnsak Thongsornkleeb: Resources, Methodology. Somsak Ruchirawat: Resources, Methodology. Onrapak Reamtong: Writing – review & editing, Writing – original draft, Supervision, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that there is no conflict of interest.

Acknowledgments

We sincerely thank Mahidol Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University for equipment support.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpddr.2024.100561.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.xlsx (31.6KB, xlsx)

References

  1. Aksenov N.A., Aksenov A.V., Kornienko A., De Carvalho A., Mathieu V., Aksenov D.A., Ovcharov S.N., Griaznov G.D., Rubin M. A nitroalkane-based approach to one-pot three-component synthesis of isocryptolepine and its analogs with potent anti-cancer activities. RSC Adv. 2018;8(64):36980–36986. doi: 10.1039/c8ra08155g. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ankarklev J., Jerlström-Hultqvist J., Ringqvist E., Troell K., Svärd S.G. Behind the smile: cell biology and disease mechanisms of Giardia species. Nat. Rev. Microbiol. 2010;8(6):413–422. doi: 10.1038/nrmicro2317. [DOI] [PubMed] [Google Scholar]
  3. Aroonkit P., Thongsornkleeb C., Tummatorn J., Krajangsri S., Mungthin M., Ruchirawat S. Synthesis of isocryptolepine analogues and their structure-activity relationship studies as antiplasmodial and antiproliferative agents. Eur. J. Med. Chem. 2015;94:56–62. doi: 10.1016/j.ejmech.2015.02.047. [DOI] [PubMed] [Google Scholar]
  4. Arruda-Costa N., Escrivani D., Almeida-Amaral E.E., Meyer-Fernandes J.R., Rossi-Bergmann B. Anti-parasitic effect of the diuretic and Na+-ATPAse inhibitor furosemide in cutaneous leishmaniasis. Parasitology. 2017;144(10):1375–1383. doi: 10.1017/S0031182017000695. [DOI] [PubMed] [Google Scholar]
  5. Bahadur V., Mastronicola D., Tiwari Hemandra K., Kumar Y., Falabella M., Pucillo Leopoldo P., Sarti P., Giuffrè A., Singh Brajendra K. O(2)-dependent efficacy of novel piperidine- and piperazine-based chalcones against the human parasite Giardia intestinalis. Antimicrob. Agents Chemother. 2014;58(1):543–549. doi: 10.1128/AAC.00990-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Benchimol M., Gadelha A.P., de Souza W. Unusual cell structures and organelles in Giardia intestinalis and Trichomonas vaginalis are potential drug targets. Microorganisms. 2022;10(11):2176. doi: 10.3390/microorganisms10112176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Campanati L., Monteiro-Leal L.H. The effects of the antiprotozoal drugs metronidazole and furazolidone on trophozoites of Giardia lamblia (P1 strain) Parasitol. Res. 2002;88(1):80–85. doi: 10.1007/s004360100502. [DOI] [PubMed] [Google Scholar]
  8. Colina-Vegas L., Godinho J.L.P., Coutinho T., Correa R.S., Souza W., Rodrigues J.C.F., Azevedo B., Navarro M. Antiparasitic activity and ultrastructural alterations provoked by organoruthenium complexes against Leishmania amazonensis. New J. Chem. 2019;43:1431–1439. [Google Scholar]
  9. Das S., Reiner D.S., Zenian J., Hogan D.L., Koss M.A., Wang C.S., Gillin F.D. Killing of Giardia lamblia trophozoites by human intestinal fluid in vitro. J. Infect. Dis. 1988;157(6):1257–1260. doi: 10.1093/infdis/157.6.1257. [DOI] [PubMed] [Google Scholar]
  10. Déchamps S., Shastri S., Wengelnik K., Vial H.J. Glycerophospholipid acquisition in Plasmodium - a puzzling assembly of biosynthetic pathways. Int. J. Parasitol. 2010;40(12):1347–1365. doi: 10.1016/j.ijpara.2010.05.008. [DOI] [PubMed] [Google Scholar]
  11. Einarsson E., Ma’ayeh S., Svärd S.G. An up-date on Giardia and giardiasis. Curr. Opin. Microbiol. 2016;34:47–52. doi: 10.1016/j.mib.2016.07.019. [DOI] [PubMed] [Google Scholar]
  12. Fava N.M., Soares R.M., Scalia L.A.M., Rodrigues da Cunha M.J., Faria E.S.M., Cury M.C. Molecular typing of canine Giardia duodenalis isolates from Minas Gerais, Brazil. Exp. Parasitol. 2016;161:1–5. doi: 10.1016/j.exppara.2015.12.003. [DOI] [PubMed] [Google Scholar]
  13. Fink M.Y., Shapiro D., Singer S.M. Giardia lamblia: laboratory maintenance, lifecycle induction, and infection of murine models. Curr Protoc Microbiol. 2020;57(1) doi: 10.1002/cpmc.102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. González-Bulnes P., Bobenchik A.M., Augagneur Y., Cerdan R., Vial H.J., Llebaria A., Ben Mamoun C. PG12, a phospholipid analog with potent antimalarial activity, inhibits Plasmodium falciparum CTP:phosphocholine cytidylyltransferase activity. J. Biol. Chem. 2011;286(33):28940–28947. doi: 10.1074/jbc.M111.268946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hajare S.T., Chekol Y., Chauhan N.M. Assessment of prevalence of Giardia lamblia infection and its associated factors among government elementary school children from Sidama zone, SNNPR, Ethiopia. PLoS One. 2022;17(3) doi: 10.1371/journal.pone.0264812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. He T.Y., Li Y.T., Liu Z.D., Cheng H., Bao Y.F., Zhang J.L. Lipid metabolism: the potential targets for toxoplasmosis treatment. Parasites Vectors. 2024;17(1):111. doi: 10.1186/s13071-024-06213-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Janik A., Perlińska-Lenart U., Gawarecka K., Augustyniak J., Bratek-Gerej E., Bernat P., Piłsyk S., Skalmowska P., Palamarczyk G., Swiezewska E., Kruszewska J.S. Synthesis of dolichols in Candida albicans is Co-regulated with elongation of fatty acids. Int. J. Mol. Sci. 2021;23(1):409. doi: 10.3390/ijms23010409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Júlio C., Vilares A., Oleastro M., Ferreira I., Gomes S., Monteiro L., Nunes B., Tenreiro R., Angelo H. Prevalence and risk factors for Giardia duodenalis infection among children: a case study in Portugal. Parasites Vectors. 2012;5:22. doi: 10.1186/1756-3305-5-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Keister D.B. Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans. R. Soc. Trop. Med. Hyg. 1983;77(4):487–488. doi: 10.1016/0035-9203(83)90120-7. [DOI] [PubMed] [Google Scholar]
  20. Kerru N., Gummidi L., Maddila S., Gangu K.K., Jonnalagadda S.B. A review on recent advances in nitrogen-containing molecules and their biological applications. Molecules. 2020;25(8):1909. doi: 10.3390/molecules25081909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kharb R., Sharma P.C., Yar M.S. Pharmacological significance of triazole scaffold. J. Enzym. Inhib. Med. Chem. 2011;26(1):1–21. doi: 10.3109/14756360903524304. [DOI] [PubMed] [Google Scholar]
  22. Kohlwein S.D., Eder S., Oh C.S., Martin C.E., Gable K., Bacikova D., Dunn T. Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae. Mol. Cell Biol. 2001;21(1):109–125. doi: 10.1128/MCB.21.1.109-125.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kraus G.A., Guo H. A direct synthesis of neocryptolepine and isocryptolepine. Tetrahedron Lett. 2010;51(31):4137–4139. [Google Scholar]
  24. Krumrie S., Capewell P., Smith-Palmer A., Mellor D., Weir W., Alexander C.L. A scoping review of risk factors and transmission routes associated with human giardiasis outbreaks in high-income settings. Curr Res Parasitol Vector Borne Dis. 2022;2 doi: 10.1016/j.crpvbd.2022.100084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lalle M., Hanevik K. Treatment-refractory giardiasis: challenges and solutions. Infect. Drug Resist. 2018;11:1921–1933. doi: 10.2147/IDR.S141468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Leung A.K.C., Leung A.A.M., Wong A.H.C., Sergi C.M., Kam J.K.M. Giardiasis: an overview. Recent Pat. Inflamm. Allergy Drug Discov. 2019;13(2):134–143. doi: 10.2174/1872213X13666190618124901. [DOI] [PubMed] [Google Scholar]
  27. Li J.C., Wang R.X., Sun Y., Zhu J.K., Hu G.F., Wang Y.L., Zhou R., Zhao Z.M., Liu Y.Q., Peng J.W., Yan Y.F., Shang X.F. Design, synthesis and antifungal activity evaluation of isocryptolepine derivatives. Bioorg. Chem. 2019;92 doi: 10.1016/j.bioorg.2019.103266. [DOI] [PubMed] [Google Scholar]
  28. Li W., Li P., Zhou X., Situ J., Lin Y., Qiu J., Yuan Y., Xi P., Jiang Z., Kong G. A cytochrome B(5)-like heme/steroid binding domain protein, PlCB5L1, regulates mycelial growth, pathogenicity and oxidative stress tolerance in Peronophythora litchii. Front. Plant Sci. 2021;12 doi: 10.3389/fpls.2021.783438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Magoulas G.E., Afroudakis P., Georgikopoulou K., Roussaki M., Borsari C., Fotopoulou T., Santarem N., Barrias E., Tejera Nevado P., Hachenberg J., Bifeld E., Ellinger B., Kuzikov M., Fragiadaki I., Scoulica E., Clos J., Gul S., Costi M.P., de Souza W., Prousis K.C., Cordeiro da Silva A., Calogeropoulou T. Design, synthesis and antiparasitic evaluation of click phospholipids. Molecules. 2021;26(14):4204. doi: 10.3390/molecules26144204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mangmee S., Adisakwattana P., Tipthara P., Simanon N., Sonthayanon P., Reamtong O. Lipid profile of Trichinella papuae muscle-stage larvae. Sci. Rep. 2020;10(1) doi: 10.1038/s41598-020-67297-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Matias A.C., Pedroso N., Teodoro N., Marinho H.S., Antunes F., Nogueira J.M., Herrero E., Cyrne L. Down-regulation of fatty acid synthase increases the resistance of Saccharomyces cerevisiae cells to H2O2. Free Radic. Biol. Med. 2007;43(10):1458–1465. doi: 10.1016/j.freeradbiomed.2007.08.003. [DOI] [PubMed] [Google Scholar]
  32. Meade J.C. P-Type transport ATPases in Leishmania and trypanosoma. Parasite. 2019;26:69. doi: 10.1051/parasite/2019069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mørch K., Hanevik K. Giardiasis treatment: an update with a focus on refractory disease. Curr. Opin. Infect. Dis. 2020;33(5):355–364. doi: 10.1097/QCO.0000000000000668. [DOI] [PubMed] [Google Scholar]
  34. Muñiz-Hernández S., Luna-Nophal A., León C.T.G., Domínguez-Ramírez L., Patrón-Soberano O.A., Nava-Castro K.E., Ostoa-Saloma P., Morales-Montor J. Dehydroepiandrosterone effect on Toxoplasma gondii: molecular mechanisms associated to parasite death. Microorganisms. 2021;9(3):513. doi: 10.3390/microorganisms9030513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Niyomploy P., Mangmee S., Tipthara P., Saeung A., Reamtong O., Sangvanich P. Lipid biomarker profiling of adult Brugia malayi using mass spectrometry detection. 3 Biotech. 2022;12(9):226. doi: 10.1007/s13205-022-03296-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nuthakki V.K., Mudududdla R., Bharate S.B. Role of basic aminoalkyl chains in the lead optimization of Indoloquinoline alkaloids. Eur. J. Med. Chem. 2022;227 doi: 10.1016/j.ejmech.2021.113938. [DOI] [PubMed] [Google Scholar]
  37. Park M.T., Kang J.A., Choi J.A., Kang C.M., Kim T.H., Bae S., Kang S., Kim S., Choi W.I., Cho C.K., Chung H.Y., Lee Y.S., Lee S.J. Phytosphingosine induces apoptotic cell death via caspase 8 activation and Bax translocation in human cancer cells. Clin. Cancer Res. 2003;9(2):878–885. [PubMed] [Google Scholar]
  38. Pintong A.R., Ruangsittichai J., Ampawong S., Thima K., Sriwichai P., Komalamisra N., Popruk S. Efficacy of Ageratum conyzoides extracts against Giardia duodenalis trophozoites: an experimental study. BMC Complement Med Ther. 2020;20(1):63. doi: 10.1186/s12906-020-2860-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Popruk S., Abu A., Ampawong S., Thiangtrongjit T., Tipthara P., Tarning J., Sreesai S., Reamtong O. Mass spectrometry-based metabolomics revealed effects of metronidazole on Giardia duodenalis. Pharmaceuticals. 2023;16(3):408. doi: 10.3390/ph16030408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pousset J.L., Martin M.T., Jossang A., Bodo B. Isocryptolepine from Cryptolepis sanguinolenta. Phytochemistry. 1995;39:735e736. [Google Scholar]
  41. Rayan P., Stenzel D., McDonnell P.A. The effects of saturated fatty acids on Giardia duodenalis trophozoites in vitro. Parasitol. Res. 2005;97(3):191–200. doi: 10.1007/s00436-005-1432-5. Epub 2005 Jul 1. [DOI] [PubMed] [Google Scholar]
  42. Reiner D.S., Wang C.S., Gillin F.D. Human milk kills Giardia lamblia by generating toxic lipolytic products. J. Infect. Dis. 1986;154(5):825–832. doi: 10.1093/infdis/154.5.825. [DOI] [PubMed] [Google Scholar]
  43. Riches A., Hart C.J.S., Trenholme K.R., Skinner-Adams T.S. Anti-giardia drug discovery: current status and gut feelings. J. Med. Chem. 2020;63(22):13330–13354. doi: 10.1021/acs.jmedchem.0c00910. [DOI] [PubMed] [Google Scholar]
  44. Riou J.F., Helissey P., Grondard L., Giorgi-Renault S. Inhibition of eukaryotic DNA topoisomerase I and II activities by indoloquinolinedione derivatives. Mol. Pharmacol. 1991;40(5):699–706. [PubMed] [Google Scholar]
  45. Rodphon W., Laohapaisan P., Supantanapong N., Reamtong O., Ngiwsara L., Lirdprapamongkol K., Thongsornkleeb C., Khunnawutmanotham N., Tummatorn J., Svasti J., Ruchirawat S. Synthesis of isocryptolepine-triazole adducts and evaluation of their cytotoxic activity. ChemMedChem. 2021;16(24):3750–3762. doi: 10.1002/cmdc.202100554. [DOI] [PubMed] [Google Scholar]
  46. Rodríguez-Morales A.J., Granados-Álvarez S., Escudero-Quintero H., Vera-Polania F., Mondragon-Cardona A., Díaz-Quijano F.A., Sosa-Valencia L., Lozada-Riascos C.O., Escobedo A.A., Liseth O., Haque U. Estimating and mapping the incidence of giardiasis in Colombia, 2009-2013. Int. J. Infect. Dis. 2016;49:204–209. doi: 10.1016/j.ijid.2016.06.005. [DOI] [PubMed] [Google Scholar]
  47. Rujimongkon K., Mungthin M., Tummatorn J., Ampawong S., Adisakwattana P., Boonyuen U., Reamtong O. Proteomic analysis of Plasmodium falciparum response to isocryptolepine derivative. PLoS One. 2019;14(8) doi: 10.1371/journal.pone.0220871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sinha S., Radotra B.D., Medhi B., Batovska D.I., Markova N., Sehgal R. Ultrastructural alterations in Plasmodium falciparum induced by chalcone derivatives. BMC Res. Notes. 2020;13(1):290. doi: 10.1186/s13104-020-05132-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Srivastava A.K., Walter R.D., Jaffe J.J. Synthetic pathways of glycerophospholipids in adult Brugia pahangi and Brugia patei. Int. J. Parasitol. 1987;17(7):1321–1328. doi: 10.1016/0020-7519(87)90098-1. [DOI] [PubMed] [Google Scholar]
  50. Vandenbosch D., Bink A., Govaert G., Cammue B.P., Nelis H.J., Thevissen K., Coenye T. Phytosphingosine-1-phosphate is a signaling molecule involved in miconazole resistance in sessile Candida albicans cells. Antimicrob. Agents Chemother. 2012;56(5):2290–2294. doi: 10.1128/AAC.05106-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Vial H.J., Eldin P., Tielens A.G., van Hellemond J.J. Phospholipids in parasitic protozoa. Mol. Biochem. Parasitol. 2003;126(2):143–154. doi: 10.1016/s0166-6851(02)00281-5. [DOI] [PubMed] [Google Scholar]
  52. Whittell L.R., Batty K.T., Wong R.P., Bolitho E.M., Fox S.A., Davis T.M., Murray P.E. Synthesis and antimalarial evaluation of novel isocryptolepine derivatives. Bioorg. Med. Chem. 2011;19(24):7519–7525. doi: 10.1016/j.bmc.2011.10.037. [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

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Data Availability Statement

Data can be provided upon request.

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