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1.
Int J Biol Macromol ; 193(Pt B): 1379-1390, 2021 Dec 15.
Article in English | MEDLINE | ID: mdl-34774593

ABSTRACT

Lymphatic filariasis and its associated health hazards have taken enormous tolls especially in the tropical and sub-tropical countries round the globe. Our present work contemplates the immunomodulatory role of filarial Thioredoxin reductase (TrxR) for the survival of the parasite inside the human host. For this, the protein TrxR was purified from the filarial parasite Setaria cervi and further substantiated through specific anti-TrxR antibody raised in mice. Both commercially available anti-TrxR antibody and laboratory raised antibody produced a single band with a molecular mass of ~80 kDa on western blot. The protein is optimally active at pH 7.0 and at temperature 37 °C. This protein contains both alpha helix and beta pleated sheet with selenocysteine at its active site. The Km was found to be 2.75 ± 0.49 mM. TrxR was found to downregulate lipopolysaccharide (LPS)-induced inflammation in macrophages due to inhibition of TLR4-NF-κB pathway. The result was further supported by the downregulation of inflammasome pathway and activation of alternatively activated macrophages upon TrxR treatment. Hence this study projects insights into the importance of filarial TrxR in host-parasite interface as well as it illustrates novel therapeutic strategy towards anti-filarial drug development.


Subject(s)
Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacology , Inflammation/drug therapy , Inflammation/metabolism , Macrophages/metabolism , Thioredoxin-Disulfide Reductase/metabolism , Thioredoxin-Disulfide Reductase/pharmacology , Animals , Cell Line , Down-Regulation/drug effects , Helminth Proteins/metabolism , Inflammation/chemically induced , Lipopolysaccharides/pharmacology , Mice , NF-kappa B/metabolism , Protein Conformation, alpha-Helical/drug effects , Protein Conformation, beta-Strand/drug effects , RAW 264.7 Cells , Setaria Nematode/drug effects , Toll-Like Receptor 4/metabolism
2.
Mol Biochem Parasitol ; 242: 111364, 2021 03.
Article in English | MEDLINE | ID: mdl-33639230

ABSTRACT

Inhibition of an imperative antioxidant enzyme with subsequent death is a victorious and widely accepted strategy to combat various infectious diseases. Among different antioxidant enzymes, thioredoxin reductase (TrxR) is an exclusive one. Studies have revealed that direct inhibition of TrxR by different classes of chemical moieties promptly results in the death of an organism. Especially the structural as well as biochemical modifications of the enzyme upon inhibition project serious threat towards the subject organism. Herein, an attempt was made to inhibit TrxR of filarial species by administering Auranofin, 1 chloro 2,4 dinitrobenzene (CDNB), Curcumin, and a novel carbamo dithioperoxo(thioate) derivative (4a). Our study has revealed that inhibition of TrxR resulted in the induction of the classical CED pathway of apoptosis along with the intrinsic and extrinsic pathways of apoptosis (Caspase mediated) routed through the ASK-1/p38 axis. Druggability analysis of filarial TrxR for the selected compounds was performed in silico through molecular docking studies. Therefore, this study attempts to decipher the mechanism of apoptosis induction following TrxR inhibition. The safety of those four compounds in terms of dose and toxicity was taken under consideration. Thitherto, the mechanism of TrxR mediated initiation of cell death in filarial parasite has remained undercover, and therefore, it is a maiden report on the characterization of apoptosis induction upon TrxR inhibition which will eventually help in generating effective antifilarial drugs in the future.


Subject(s)
Anthelmintics/pharmacology , Auranofin/pharmacology , Caspases/genetics , Curcumin/pharmacology , Dinitrochlorobenzene/pharmacology , Setaria Nematode/drug effects , Thioredoxin-Disulfide Reductase/antagonists & inhibitors , Animals , Anthelmintics/chemistry , Apoptosis/drug effects , Apoptosis/genetics , Auranofin/chemistry , Binding Sites , Caspases/metabolism , Cattle , Curcumin/chemistry , Dinitrochlorobenzene/chemistry , Gene Expression Regulation , Helminth Proteins/genetics , Helminth Proteins/metabolism , MAP Kinase Kinase Kinase 5/genetics , MAP Kinase Kinase Kinase 5/metabolism , Microfilariae/drug effects , Microfilariae/enzymology , Microfilariae/growth & development , Models, Molecular , Oxidative Stress , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Reactive Oxygen Species/agonists , Reactive Oxygen Species/metabolism , Setaria Nematode/enzymology , Setaria Nematode/growth & development , Signal Transduction , Thioredoxin-Disulfide Reductase/chemistry , Thioredoxin-Disulfide Reductase/genetics , Thioredoxin-Disulfide Reductase/metabolism , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism
3.
Vet Parasitol ; 290: 109357, 2021 Feb.
Article in English | MEDLINE | ID: mdl-33516120

ABSTRACT

In this ex vivo study, S. cervi parasitoses were treated with Ivermectin (50 µM), Albendazole (200 µM) alone and Ivermectin + Albendazole (50 + 200 µM) at 37°C for 8 h and the motility and viability of the parasitoses were evaluated. Individually both drugs Ivermectin (Iver) and Albendazole (Alb) are reported to affect the function and integrity of ER, however till date, no reports are available on the functional changes in ER due to a combined Iver and Alb treatment of bovine helminth parasitosis. Here, we report the lethal effect of a combination treatment of Iver and Alb against adult bovine filarial parasitosis Setaria cervi. The underlying mechanism of drug action was elucidated by performing a systematic biochemical, molecular and proteomics based study. Altered calcium homeostasis in drug treated parasitoses lead to reduction in levels of total Endoplasmic Reticulum (ER) calcium by 50 % and 61 % and elevation by 50 % and 63 % in cytosol in Iver alone and Iver + Alb treated parasitoses respectively. Further, it was found that upregulated expression of ER localized GRP94, galactosyltransferase and glycosyltransferase activity in addition to reduction in activity of PDI indicated ER stress mechanisms being operative under combined drug treatment. Marked rise of 79 % reactive oxygen species and reduced antioxidant levels induced oxidative stress in drug treated parasitosis. The collective effect of both ER and oxidative stress might have triggered apoptosis, as evidenced by the elevated calpain activity, reduction of 67 % in cytochrome c oxidase and 83 % rise in caspase-3 activity in the Iver + Alb treated parasitoses respectively. The ER proteome analysis by 2D gel electrophoresis revealed 76 spots in the control and 56 spots in the treated proteome. A MALDI-MS/MS analysis of some of the differentially expressed spots of the combination drug treated parasitoses identified glucuronosyltransferase as a major upregulated protein with a fold change of 1.81. Trafficking protein, acyl transferase, MATH involved in protein folding were also found to be downregulated. Thus, this study based on biochemical and proteomic approaches indicates that a combination of anti-filarial drugs Iver and Alb can alter calcium homeostasis in bovine filarial parasitosis leading to induction of ER stress culminating into apoptosis.


Subject(s)
Albendazole/pharmacology , Endoplasmic Reticulum Stress/drug effects , Ivermectin/pharmacology , Setaria Nematode/drug effects , Signal Transduction/drug effects , Albendazole/administration & dosage , Animals , Antiparasitic Agents/administration & dosage , Antiparasitic Agents/pharmacology , Biomarkers , Drug Therapy, Combination , Female , Ivermectin/administration & dosage , Motor Activity/drug effects , Oxidative Stress
4.
Naunyn Schmiedebergs Arch Pharmacol ; 393(8): 1341-1356, 2020 08.
Article in English | MEDLINE | ID: mdl-32002575

ABSTRACT

The aim of the present study was to assess the effect of diethylcarbamazine (DEC), siver nanoparticles (AgNPs), nitazoxanide (NTZ), and a combination of nitazoxanide with silver nanoparticle (NTZ+AgNPs) against the microfilariae of Setaria cervi in experimentally infected albino rats. The NTZ+AgNPs was synthesized and subsequently characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV-visible absorption Spectra (UV-VIS), Fourier transforms infrared spectroscopy (FTIR), and energy dispersive X-ray (EDX) spectra. Twenty male albino rats were divided into 5 groups. Groups I, II, III, and IV were treated with DEC, AgNPs, NTZ, and NTZ+AgNPs, while group V was taken as untreated infected control. After the establishment of infection, microfilaraemic rats were treated with aforesaid drugs for 6 days at 100 mg/kg body weight. Efficacy of drugs was observed by counting the microfilariae in the blood of albino rats every 3rd day till microfilariae disappeared. Blood was taken at every 10 days interval till 40 days for biochemical studies to assess the level of antioxidant enzymes. NTZ+AgNPs proved to be the most effective drug which cleared the microfilariae within 18 days of infection when compared with DEC, AgNPs and NTZ where microfilariae persisted up to 24, 36, and 33 days, respectively. Oxidative stress is common inflammatory process associated with many diseases including filariasis. An enhanced antioxidant activity of NTZ+AgNPs was observed in the infected rats which was evident by quick disappearance of microfilariae due to increased oxidative stress. It clearly indicated positive contribution of the NTZ+AgNPs to the host together with harmful effect on the parasite. Hence, AgNPs improved the NTZ efficacy against S. cervi infection in albino rats and proved as a successful synergistic combination.


Subject(s)
Filaricides/pharmacology , Metal Nanoparticles , Nanocomposites , Nitro Compounds/pharmacology , Setaria Nematode/drug effects , Setariasis/drug therapy , Silver/pharmacology , Thiazoles/pharmacology , Animals , Diethylcarbamazine/pharmacology , Disease Models, Animal , Drug Compounding , Drug Synergism , Filaricides/administration & dosage , Host-Parasite Interactions , Male , Nitro Compounds/administration & dosage , Rats , Setaria Nematode/growth & development , Setaria Nematode/metabolism , Setariasis/parasitology , Silver/administration & dosage , Thiazoles/administration & dosage
5.
Parasitology ; 146(11): 1451-1461, 2019 09.
Article in English | MEDLINE | ID: mdl-31104638

ABSTRACT

The present work aims to explore the mechanism of action of C-cinnamoyl glycoside as an antifilarial agent against the bovine filarial nematode Setaria cervi. Both apoptosis and autophagy programmed cell death pathways play a significant role in parasitic death. The generation of reactive oxygen species, alteration of the level of antioxidant components and disruption of mitochondrial membrane potential may be the causative factors that drive the parasitic death. Monitoring of autophagic flux via the formation of autophagosome and autophagolysosome was detected via CYTO ID dye. The expression profiling of both apoptotic and autophagic marker proteins strongly support the initial findings of these two cell death processes. The increased interaction of pro-autophagic protein Beclin1 with BCL-2 may promote apoptotic pathway by suppressing anti-apoptotic protein BCL-2 from its function. This in turn partially restrains the autophagic pathway by engaging Beclin1 in the complex. But overall positive increment in autophagic flux was observed. Dynamic interaction and regulative balance of these two critical cellular pathways play a decisive role in controlling disease pathogenesis. Therefore, the present experimental work may prosper the chance for C-cinnamoyl glycosides to become a potential antifilarial therapeutic in the upcoming day after detail in vivo study and proper clinical trial.


Subject(s)
Apoptosis/drug effects , Autophagy/drug effects , Filaricides/pharmacology , Glycosides/pharmacology , Setaria Nematode/drug effects , Wuchereria bancrofti/drug effects , Animals , Setaria Nematode/physiology , Wuchereria bancrofti/physiology
6.
J Helminthol ; 93(5): 519-528, 2019 Sep.
Article in English | MEDLINE | ID: mdl-30032733

ABSTRACT

Efficacious therapeutic strategies against lymphatic filariasis are always sought after. However, natural products are a promising resource for developing effective antifilarial agents. Azadirachtin, a significant tetranortriterpenoid phytocompound found in Azadirachta indica, was evaluated in vitro for antifilarial potential against the filarial parasite Setaria cervi. Dye exclusion and MTT assay confirmed the antifilarial potential of azadirachtin against S. cervi with a median lethal dose (LC50) of 6.28 µg/ml for microfilariae (mf), and 9.55 µg/ml for adult parasites. Morphological aberrations were prominent in the histological sections of the azadirachtin-exposed parasites. Moreover, alterations in the reactive oxygen species (ROS) parameters in treated parasites were evident. Induction of apoptosis in treated parasites was confirmed by DNA laddering, acridine orange (AO)/ethidium bromide (EtBr) double staining and in situ DNA fragmentation. The downregulation of anti-apoptotic CED-9 and upregulation of proapoptotic EGL-1, CED-4 and CED-3 at both the transcription and translation levels confirmed apoptosis execution at the molecular level. Changes in the gene expressions of nuc-1, cps-6 and crn-1 further clarified the molecular cause of DNA degradation. Furthermore, azadirachtin was found to be non-toxic in both in vitro and in vivo toxicity analyses. Therefore, the experimental evidence detailed the pharmacological effectiveness of azadirachtin as a possible therapeutic agent against filariasis.


Subject(s)
Anthelmintics/pharmacology , Apoptosis , Limonins/pharmacology , Plant Extracts/pharmacology , Reactive Oxygen Species/metabolism , Setaria Nematode/drug effects , Animals , DNA Fragmentation , Elephantiasis, Filarial/drug therapy , Female , Helminth Proteins/genetics , Lethal Dose 50 , Male , Setaria Nematode/genetics
7.
PLoS One ; 13(12): e0208201, 2018.
Article in English | MEDLINE | ID: mdl-30521546

ABSTRACT

Development of antifilarial drug from the natural sources is considered as one of the most efficacious, safe, and affordable approaches. In this study, we report the antifilarial activity of a leguminous plant Cajanus scarabaeoides (L.) Thouars. The polyphenol-rich ethanolic extract obtained from the stem part of the plant C. scarabaeoides (EECs) was found to be efficient in killing the filarial nematode Setaria cervi in all the three developmental stages viz. oocytes, microfilariae (Mf) and adults with LD50 values of 2.5, 10 and 35 µg/ml, respectively. While studying the molecular mechanism of action, we found that induction of oxidative stress plays the key role in inducing the mortality in S. cervi. The redox imbalance finally results in activation of the nematode CED pathway that executes the death of the parasite. Intriguingly, EECs was found to be selectively active against the worm and absolutely non-toxic to the mammalian cells and tissues. Taken together, our experimental data demonstrate that C. scarabaeoides can be chosen as an affordable natural therapeutic for treating filarial infection in the future with high efficacy and less toxicity.


Subject(s)
Cajanus/chemistry , Filaricides/pharmacology , Plant Extracts/pharmacology , Setaria Nematode/drug effects , Animals , Apoptosis/drug effects , Cattle , Ethanol/chemistry , Female , Filaricides/chemistry , Filaricides/isolation & purification , Filaricides/therapeutic use , Lethal Dose 50 , Models, Animal , Oxidative Stress/drug effects , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Extracts/therapeutic use , Plant Stems/chemistry , Polyphenols/isolation & purification , Polyphenols/pharmacology , Reactive Oxygen Species/metabolism , Setariasis/drug therapy
8.
Sci Rep ; 8(1): 12073, 2018 08 13.
Article in English | MEDLINE | ID: mdl-30104608

ABSTRACT

Search of potent antifilarial drugs has been a major thrust area in tropical medicine research over the decades. Herein, we report 4,7-dimethyl-3,4,7,8-tetrahydro-3λ6-[1,2]thiazino[4,3-f]quinoline-3,3,8-trione (8l) as a new class of antifilarial agent which is extremely potent, with lethality against all the developmental stages (oocyte, microfilaria and adult) of the filarial parasite Setaria cervi. Molecular investigation on its mode of action revealed that 8l is a typical inducer of reactive oxygen species that triggers oxidative stress inside the filarid and further signals induction of apoptosis by activating both intrinsic and extrinsic pathways. Moreover, 8l is also active against Wolbachia, the essential endosymbiont of several human infectious filarids. Selective toxicity against filarial parasites and non-toxic nature in rat model were found as unique traits of 8l to be a future medicine. Taken en masse, this maiden report on a novel quinolone fused cyclic sulfonamide presents a promising therapeutic lead for lymphatic filariasis in future.


Subject(s)
Elephantiasis, Filarial/drug therapy , Filaricides/pharmacology , Quinolones/pharmacology , Setaria Nematode/drug effects , Sulfonamides/pharmacology , Aedes , Animals , Apoptosis/drug effects , Cattle , Disease Models, Animal , Elephantiasis, Filarial/parasitology , Female , Filaricides/chemistry , Filaricides/therapeutic use , Humans , Male , Mice , Oxidative Stress/drug effects , Parasitic Sensitivity Tests , Quinolones/chemistry , Quinolones/therapeutic use , RAW 264.7 Cells , Rats , Reactive Oxygen Species/metabolism , Setaria Nematode/metabolism , Sulfonamides/chemistry , Sulfonamides/therapeutic use , Wolbachia/drug effects , Wolbachia/metabolism
9.
Colloids Surf B Biointerfaces ; 161: 236-243, 2018 Jan 01.
Article in English | MEDLINE | ID: mdl-29080508

ABSTRACT

The present work seeks to explore the antifilarial activity of biopolymer functionalized gold nanoparticles (AuNPs) against human filarial parasite (Wuchereria bancrofti) through Nrf2 signaling for the first time. A natural polymer, chitosan is used along with Terminalia chebula extract to synthesize AuNPs following the principles of green chemistry. The probable mode of action of AuNPs as filaricidal agent has been investigated in detail using model filarial parasite, Setaria cervi (bovine parasite). Biopolymers inspired AuNPs exhibit superior antifilarial activity against both human and bovine filarial parasites, and are able to induce oxidative stress and apoptotic cell death in filarial parasites mediated through mitochondria. AuNPs also alter the Nrf2 signaling. In addition, the synthesized nanomaterials appear to be nontoxic to mammalian system. Thus the present mechanistic study, targeting human filarial parasites, has the potential to increase the therapeutic prospects of AuNPs to control lymphatic filariasis in the upcoming days.


Subject(s)
Filaricides/chemistry , Gold/chemistry , Metal Nanoparticles/chemistry , Nanomedicine/methods , Animals , Apoptosis/drug effects , Cattle , Chitosan/chemistry , Filariasis/drug therapy , Filariasis/parasitology , Filaricides/administration & dosage , Gold/administration & dosage , Humans , Metal Nanoparticles/administration & dosage , Plant Extracts/chemistry , Rats, Wistar , Setaria Nematode/drug effects , Setaria Nematode/physiology , Terminalia/chemistry , Wuchereria bancrofti/drug effects , Wuchereria bancrofti/physiology
10.
Exp Parasitol ; 177: 13-21, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28351683

ABSTRACT

Absence of a drug that kills adult filarial parasites remains the major challenge in eliminating human lymphatic filariasis (LF); the second leading cause of long-term and permanent disability. Thus, the discovery of novel antifilarial natural products with potent adulticidal activity is an urgent need. In the present study, methanol extracts of leaves, bark and winged seeds of Dipterocarpus zeylanicus (Dipterocarpaceae) were investigated for macro and microfilaricidal activity. Two antifilarial triterpene saponins were isolated from winged seed extracts by bioactivity guided chromatographic separation and identified using Nuclear Magnetic Resonance and mass spectroscopic analysis as oleanolic acid 3-O-ß-D- glucopyranoside (1) (IC50 = 20.54 µM for adult worms, 19.71 µM for microfilariae ) and oleanolic acid 3-O-α-L-arabinopyranoside (2) (IC50 = 29.02 µM for adult worms, 25.99 µM for microfilariae). Acid hydrolysis of both compounds yielded oleanolic acid (3) which was non or least toxic to human peripheral blood mono nuclear cells (Selectivity index = >10) while retaining similar macrofilaricidal (IC50 = 38.4 µM) and microfilaricidal (IC50 = 35.6 µM) activities. In adult female worms treated with 50 and 100 µM doses of oleanolic acid, condensation of nuclear DNA, apoptotic body formation and tissue damage was observed by using Hoechst 33342 staining, TUNEL assay and Hematoxylin and Eosin staining respectively. A dose dependent increase in caspase 3/CED3 activity and decrease in total protein content were also observed in these parasites. A dose dependant DNA fragmentation was observed in adult parasites and microfilariae. Decreased levels of reduced glutathione (GSH) and elevated levels of glutathione S transferase (GST), superoxide dismutase (SOD) and reactive oxygen species (ROS) were also observed in parasites treated with oleanolic acid indicating an oxidative stress mediated apoptotic event. Compound 3/oleanolic acid was thus identified as a potent and safe antifilarial compound in vitro.


Subject(s)
Apoptosis/drug effects , Embryophyta/chemistry , Oleanolic Acid/pharmacology , Oxidative Stress/drug effects , Saponins/chemistry , Setaria Nematode/drug effects , Animals , Caspase 3/metabolism , DNA Fragmentation/drug effects , Dose-Response Relationship, Drug , Female , In Situ Nick-End Labeling , Inhibitory Concentration 50 , Lethal Dose 50 , Oleanolic Acid/isolation & purification , Plant Bark/chemistry , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Plant Leaves/chemistry , Reactive Oxygen Species/metabolism , Seeds/chemistry , Setaria Nematode/cytology , Setaria Nematode/metabolism , Setariasis/drug therapy , Setariasis/parasitology , Staining and Labeling , Triterpenes/chemistry
11.
Carbohydr Polym ; 157: 1666-1676, 2017 Feb 10.
Article in English | MEDLINE | ID: mdl-27987881

ABSTRACT

The gold nanoparticles (AuNPs) have been synthesized biogenically by using black pepper (Piper nigrum) extract according to the principles of green chemistry in presence and absence of a biopolymer, chitosan. A comprehensive study (up to cellular level) on the antifilarial (against Setaria cervi) activity of AuNPs has been made for the first time with a view to use it clinically. The bioactivity of biopolymer capped biogenic AuNP increases significantly compared to simple biogenic AuNP. The biopolymer plays an important role in inspiring AuNP through its inherent positive charges and hydrophobicity. The developed nanomaterial boosts the production of ROS (reactive oxygen species) and misbalances the antioxidant parameters of parasites such as GSH, GST, GPx, SOD and catalase. The produced ROS ultimately induces oxidative stress, which leads to apoptotic cell death in filarial worms. The synthesized nanomaterials exhibit negligible toxicity towards human PBMCs. The present study may serve as a fruitful platform to explore biopolymer capped gold nanoparticles as efficient antifilarial therapeutics.


Subject(s)
Anthelmintics/pharmacology , Chitosan , Gold , Metal Nanoparticles , Setaria Nematode/drug effects , Animals , Biopolymers , Cells, Cultured , DNA Fragmentation , Humans
12.
Exp Parasitol ; 167: 50-60, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27174667

ABSTRACT

Human lymphatic filariasis (LF) is mainly caused by filarial parasite Wuchereria bancrofti and is the second leading cause of long term and permanent disability in tropical countries. To date, incapability to eliminate long lived adult parasites by current drugs remains the major challenge in the elimination of LF. Hence, in the current study, the efficacy of rhizome extracts of Curcuma zedoaria (a plant traditionally used in Sri Lanka in the management of LF) was evaluated as an effective filaricide in vitro. Sequential solvent extracts of C. zedoaria rhizomes were screened for in vitro antifilarial activity at 0.01-1 mg/mL concentrations by motility inhibition assay and 3-(4, 5 dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT) reduction assay using cattle parasite Setaria digitata as a model organism. Exposure of parasites to hexane and chloroform extracts of C. zedoaria caused a dose dependant reduction in motility and viability of microfilariae (IC50 = 72.42 µg/mL for hexane extract, 191.14 µg/mL for chloroform extract) and adult parasites (IC50 = 77.07 µg/mL for hexane extract, 259.87 µg/mL for chloroform extract). Both extracts were less toxic to human peripheral blood mononuclear cells when compared to filariae. A dose dependant increase in caspase 3/CED 3 and a decrease in total protein content, cyclooxygenase (COX) and protein tyrosine phosphatase (PTP) activities were observed in adult parasites treated with hexane or chloroform extract. A significant degree of chromatin condensation and apoptotic body formation were also observed in these worms by Hoechst 33342 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) staining respectively. Dose dependant chromosomal DNA laddering was observed in treated adult worms but not in microfilariae in response to both extracts. Oxidative stress parameters such as reduction in reduced glutathione (GSH) levels and increase in glutathione s transferase (GST), superoxide dismutase (SOD) and catalase activities, increased reactive oxygen levels (ROS) and lipid peroxidation were also observed indicating that an apoptotic event is induced by reactive oxygen species.


Subject(s)
Curcuma/chemistry , Plant Extracts/pharmacology , Pyroptosis/drug effects , Reactive Oxygen Species/metabolism , Rhizome/chemistry , Setaria Nematode/drug effects , Animals , Caspase 3/metabolism , Cattle , DNA Fragmentation , Dose-Response Relationship, Drug , Female , Humans , In Situ Nick-End Labeling , Inhibitory Concentration 50 , Leukocytes, Mononuclear/drug effects , Peritoneal Cavity/parasitology , Plant Extracts/toxicity , Setaria Nematode/cytology , Setaria Nematode/metabolism
13.
Free Radic Biol Med ; 93: 130-44, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26849945

ABSTRACT

Lymphatic filariasis, affecting around 120 million people in 80 countries worldwide, is an extremely painful disease and caused permanent and long term disability. Owing to its alarming prevalence there is immediate need for development of new therapeutics. A series of trans-stilbene derivatives were synthesized using aqueous reaction condition showing potential as antifilarial agents demonstrated in vitro. MTT reduction assay and dye exclusion test were performed to evaluate the micro and macrofilaricidal potential of these compounds. Amid 20 trans-stilbene derivatives together with Resveratrol (RSV), a multifunctional natural product was screened; nine compounds (28, 29, 33, 35, 36, 38, 39, 41 and 42) have showed promising micro and macrofilaricidal activities and four of them (28, 39, 41 and 42) showed better effectiveness than RSV. In the treated parasites apoptosis was established by DNA laddering, in situ DNA fragmentation and FACS analysis. The generation of ROS in the treated parasites was indicated by the depletion in the level of GSH, GR and GST activity and elevation of SOD, catalase, GPx activity and superoxide anion and H2O2 level. Along with the ROS generation and oxidative stress, the decreased expression of anti-apoptotic ced-9 gene and increased expression of nematode specific pro-apoptotic genes, egl-1, ced-4 and ced-3 at the level of transcription and translation level; the up-regulation of caspase-3 activity and involvement of caspase-8,9,3, cytochrome-c and PARP were also observed and which denotes the probable existence of both extrinsic and intrinsic pathways apoptosis in parasitic nematodes. This observation is reported first time and thus it confirmed the mode of action and effectiveness of the compounds. Further, the comparative bioavailability-pharmacokinetics studies showed that compound 28 possesses comparable properties with Ivermectin. This study will certainly intensify our understanding of the pharmacological importance of trans-stilbenes as an anti-filarial agent.


Subject(s)
Elephantiasis, Filarial/drug therapy , Oxidative Stress/drug effects , Setaria Nematode/drug effects , Stilbenes/pharmacology , Animals , Apoptosis/drug effects , Caspases/biosynthesis , Caspases/metabolism , DNA Fragmentation/drug effects , Elephantiasis, Filarial/metabolism , Elephantiasis, Filarial/parasitology , Gene Expression Regulation/drug effects , Humans , Hydrogen Peroxide/metabolism , Reactive Oxygen Species/metabolism , Resveratrol , Setaria Nematode/pathogenicity , Stilbenes/chemical synthesis , Stilbenes/chemistry
14.
Exp Parasitol ; 160: 39-48, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26627139

ABSTRACT

Green synthesis of silver nanomaterial plays a pivotal role in the growing field of nanotechnology. Development of anti-parasitic drugs from plant metabolites has been in regular practice from the ancient period but most of them were discarded due to their inefficiency to control diseases effectively. At present, nanoparticles are used for developing anti-parasitic therapy for their unique properties such as smallest in size, bio-ability, bio-compatibility and penetration capacity into a cell. The present study aims at synthesis of silver nanoparticles (AgNPs) by using funicles extract of Acacia auriculiformis and tests its efficacy as antifilarial. Experimental evidence show that AgNPs are effective at a very low concentration compared to crude plant extracts. Synthesis of these nanoparticles is a single-step, biogenic, cost effective and eco-friendly process. Synthesized nanoparticles were characterized by UV-Vis spectroscopy, TEM, SAED, FTIR, EDX, FESEM and Z-potential. The antifilarial efficacy of AgNPs was tested against different life cycle stages of bovine filarial parasite Setaria cervi by morphological study, motility assessment and viability assay. These nanoparticles are found to have antifilarial activity with LC50 of 5.61 µg/mL and LC90 of 15.54 µg/mL against microfilaria of S. cervi. The microscopic findings and the detailed molecular studies confirmed that green synthesized AgNPs were effective enough to induce apoptosis through up regulation of ROS (reactive oxygen species).


Subject(s)
Acacia/chemistry , Apoptosis/drug effects , Metal Nanoparticles/adverse effects , Reactive Oxygen Species/metabolism , Setaria Nematode/drug effects , Animals , Apoptosis/physiology , Cattle , DNA Fragmentation , DNA, Helminth/chemistry , DNA, Helminth/genetics , Female , In Situ Nick-End Labeling , Macrophages, Peritoneal/drug effects , Male , Metal Nanoparticles/administration & dosage , Metal Nanoparticles/toxicity , Microscopy, Electron, Scanning , Photoelectron Spectroscopy , Plant Extracts/chemistry , Rats , Rats, Wistar , Setaria Nematode/cytology , Setaria Nematode/genetics , Setaria Nematode/metabolism , Setariasis/parasitology , Silver , Spectroscopy, Fourier Transform Infrared , Up-Regulation
15.
Biochem Biophys Res Commun ; 458(1): 194-200, 2015 Feb 27.
Article in English | MEDLINE | ID: mdl-25645020

ABSTRACT

A 67 kDa cytosolic FERM domain containing protein having significant protein tyrosine phosphatases activity (PTPL) has been purified to homogeneity from Setaria cervi, a bovine filarial parasite. The MALDI-MS/MS analysis of the purified protein revealed 16 peptide peaks showing nearest match to Brugia malayi Moesin/ezrin/radixin homolog 1 protein and one peptide showing significant similarity with a region lying in the catalytic domain of human PTPD1. PTPL showed significant cross reactivity with the human PTP1B antibody and colocalize with actin in the coelomyrian cells of hypodermis in the parasite. PTPL was stress regulated as it showed marked decrease in the expression when exposed to Aspirin, an antifilarial drug and Phenylarsine Oxide, PTP inhibitor.


Subject(s)
Cytosol/metabolism , Helminth Proteins/chemistry , Helminth Proteins/metabolism , Protein Tyrosine Phosphatases/metabolism , Setaria Nematode/chemistry , Amino Acid Sequence , Animals , Arsenicals/pharmacology , Aspirin/pharmacology , Catalytic Domain , Cross Reactions , Female , Helminth Proteins/isolation & purification , Humans , Molecular Sequence Data , Protein Structure, Tertiary , Protein Tyrosine Phosphatases/chemistry , Sequence Homology, Amino Acid , Setaria Nematode/drug effects , Setaria Nematode/pathogenicity
16.
Pharm Biol ; 53(6): 813-23, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25720973

ABSTRACT

CONTEXT: Lymphatic filariasis is a major neglected tropical disease. Diospyros perigrena Gurke (Ebenaceae) was selected for antifilarial chemotherapy because of unavailability of proper medicine. In India, different parts of this plant were used for the treatment of diabetes, diarrhea, dysentery, cholera, mouth ulcers, and wounds. OBJECTIVE: The present study was undertaken to access antifilarial potential and mechanism of action of n-butanol extract (NBE) of D. perigrena stem bark on Setaria cervi Rudolphi (Onchocercidae). MATERIALS AND METHODS: In vitro efficacy and apoptotic mechanism were evaluated by Hoechst, TUNEL, DNA fragmentation assay, pro- and anti-apoptotic gene expression in NBE (250, 125, 62.5, 31.25, and 15.6 µg/ml)-treated S. cervi after 24 h of incubation. Reactive oxygen species (ROS) up-regulation was also determined by GSH, GST, SOD assays, and super oxide anion level. RESULTS: Significant in vitro antifilarial activity of NBE was found 50% inhibitory concentration (IC50): adult = 57.6 µg/ml, microfilariae (mf) = 56.1 µg/ml, and lethal dose (LD100) in mf is 187.17 µg/ml) after 24 h of treatment. NBF-induced apoptosis was proved by Hoechst, TUNEL, RT-PCR, and Western blot method. NBF (250 µg/ml) decreased the level of GSH (17.8%) and GST (65.4%), increased SOD activity (1.42-fold) and super oxide anion production (1.32-fold) in the treated parasite which culminated into ROS up-regulation. DISCUSSION AND CONCLUSION: NBE induced apoptosis in different life cycle stages of S. cervi. In future, a detailed study of NBF will give us a novel antifilarial compound which will be used for antifilarial chemotherapy.


Subject(s)
Apoptosis/drug effects , Diospyros/chemistry , Filaricides/pharmacology , Plant Bark/chemistry , Plant Extracts/pharmacology , Reactive Oxygen Species/metabolism , Setaria Nematode/drug effects , 1-Butanol , Animals , Bisbenzimidazole , Coloring Agents , DNA/drug effects , Filariasis/drug therapy , Filariasis/psychology , In Situ Nick-End Labeling , Setaria Nematode/metabolism , Solvents , Tetrazolium Salts , Thiazoles
17.
Parasitol Res ; 113(10): 3581-9, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25028209

ABSTRACT

The ecto protein tyrosine phosphatases (PTP) are known to play a crucial role in the pathogenesis and survival of the intracellular parasites. However, their presence and role in filarial parasites is still unknown. We found a significant amount of tyrosine phosphatase activity in the surface antigen fraction extracted from Setaria cervi (S. cervi), a bovine filarial parasite. An antibody designed against the conserved catalytic core of human protein tyrosine phosphatases, PTP1B cross reacted with a 63 kDa band in the surface antigen. We detected a significant amount of PTP activity in the intact S. cervi adult parasites as well as microfilariae in this study for the first time. This PTP may be localized on the surface of the parasite with an exposed active site available for the external substrates. The PTP activity was also inhibited by sodium orthovanadate and phenyl arsine oxide, specific inhibitors of PTP in both the life stages. The Km and Vmax for PTP in the adult parasites and microfilariae were determined to be 2.574 ± 0.14 mM; 206.3 ± 2.75 µM Pi/h/two parasites and 5.510 ± 0.59 mM; 62.27 ± 2.27 µM Pi/h/10(6) parasites respectively using O-P-L-Tyrosine as substrate. Interestingly, a positive correlation was observed between the inhibition in PTP activity and reduction in the motility/ viability of the parasites when they were subjected to the specific PTP inhibitors (Orthovanadate and Phenyl arsine oxide) for 4 h in the KRB maintenance medium. The activity was also significantly inhibited in the parasites exposed to antifilarial drug/compounds for e.g. Diethylcarbamazine, Acetylsalicylic Acid and SK7, a methyl chalcone. Therefore suggesting a possible role played by PTP in the survival of the parasite, its interaction with the host as well as in the screening of newly synthesized antifilarials/drugs.


Subject(s)
Cattle Diseases/parasitology , Filaricides/pharmacology , Protein Tyrosine Phosphatases/metabolism , Setaria Nematode/enzymology , Setariasis/parasitology , Animals , Arsenicals/pharmacology , Aspirin/pharmacology , Catalytic Domain , Cattle , Chalcone/pharmacology , Diethylcarbamazine/pharmacology , Enzyme Inhibitors/pharmacology , Female , Host-Parasite Interactions , Microfilariae , Protein Tyrosine Phosphatases/antagonists & inhibitors , Setaria Nematode/drug effects , Setaria Nematode/physiology , Vanadates/pharmacology
18.
Parasitol Int ; 63(5): 717-28, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25009078

ABSTRACT

A bio-assay guided fractionation and purification approach was used to examine in vitro antifilarial activities of the crude methanolic extract of Nyctanthes arbortristis as well as fractions and isolated compound. From ethyl-acetate fraction we isolated and identified a triterpenoid compound which has been characterized as ursolic acid (UA) by HPLC and NMR data. We are reporting for the first time isolation and identification of UA from the leaves of N. arbortristis. The crude extract and UA showed significant micro- as well as macrofilaricidal activities against the oocyte, microfilaria and adult of Setaria cervi (S. cervi) by dye exclusion test and MTT reduction assay. Significant microfilaricidal activity of UA was further proved against mf of W. bancrofti by viability assay. The findings thus provide a new lead for development of a suitable filaricide from natural products. The molecular mechanism of UA was investigated by performing TUNEL, Hoechst staining, Annexin V-Cy3, flow cytometric analysis and DNA fragmentation assay. Differential expressions of pro- and anti-apoptotic genes were observed at the transcription and translational levels in a dose-dependent manner. Depletion in the worm GSH level and elevation in the parasite GST, SOD and super oxide anion indicated the generation of ROS. In this investigation we are reporting for the first time that UA acts its antifilarial effect through induction of apoptosis and by downregulating and altering the level of some key antioxidants like GSH, GST and SOD of S. cervi.


Subject(s)
Filaricides/pharmacology , Oleaceae/chemistry , Setaria Nematode/drug effects , Triterpenes/pharmacology , Wuchereria bancrofti/drug effects , Adult , Animals , Blotting, Western , Dose-Response Relationship, Drug , Female , Filaricides/chemistry , Glutathione , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Humans , Male , Oxidative Stress/drug effects , Plant Leaves/chemistry , Plants, Medicinal , Reverse Transcriptase Polymerase Chain Reaction , Superoxide Dismutase , Superoxides , Triterpenes/chemistry , Ursolic Acid
19.
Exp Parasitol ; 136: 41-58, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24275557

ABSTRACT

Lymphatic filariasis, a global cause of morbidity needs much more attention in developing potent therapeutics that can be effective against both microfilariae (mf) and adults. Efficient botanicals that can induce apoptosis of filarial parasites possibly can provide a direction towards developing new class of antifilarials. In this work we have evaluated the antifilarial efficacy of an optimized polyphenol rich ethanolic extract of Azadirachta indica leaves (EEA). A. indica A. Juss has been widely used in the traditional Indian medicinal system 'Ayurveda' for the treatment of a variety of ailments. A thorough investigation towards biochemical and molecular mechanisms describing ROS mediated apoptosis in Setaria cervi was performed. Motility reduction, MTT reduction assay and dye exclusion test have confirmed the micro- and macrofilaricidal potential of EEA. Alterations were visible in mf and trichrome stained section of EEA-treated adult worms. We have found cellular disturbances in EEA-treated parasites characterized by chromatin condensation, in situ DNA fragmentation and nucleosomal DNA laddering. Depletion in worm GSH level and elevation in parasite GST, SOD, catalase, GPx and superoxide anion indicated the generation of ROS. Our results provided experimental evidence supporting that EEA causes a decreased expression of anti-apoptotic genes and increased pro-apoptotic gene expression at the level of both transcription and translation. Here we are reporting for the first time that antifilarial activity of EEA is mediated by ROS up regulation and apoptosis.


Subject(s)
Azadirachta/chemistry , Filaricides/pharmacology , Plant Extracts/pharmacology , Polyphenols/pharmacology , Reactive Oxygen Species/metabolism , Setaria Nematode/drug effects , Analysis of Variance , Animals , Apoptosis/genetics , Cattle , DNA Fragmentation/drug effects , Dose-Response Relationship, Drug , Female , Filaricides/isolation & purification , Gene Expression Regulation/drug effects , Ivermectin/pharmacology , Male , Oxidation-Reduction/drug effects , Plant Extracts/isolation & purification , Plant Leaves/chemistry , Polyphenols/isolation & purification , Setaria Nematode/genetics , Setaria Nematode/metabolism
20.
Asian Pac J Trop Med ; 6(9): 689-92, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23827144

ABSTRACT

OBJECTIVE: To evaluate the possible antifilarial effect of ethyl acetate extract of Vitex negundo (Verbenaceae) leaves against Setaria cervi filarial parasite in vitro. METHODS: In vitro screening was done by the method of motility inhibition and MTT reduction assay with concentrations of 0.03 to 1.00 mg/mL for 2 to 24 h incubation periods respectively, for possible antifilarial effect by comparing with control. RESULTS: In motility assay, complete inhibition of motility was observed and in MTT reduction assay which gave >50% reduction for concentrations 0.20, 0.50 and 1.00 mg/mL at 10, 6 and 2 h incubation periods respectively in a dose dependent manner (P<0.05). An antifilarial effect imparted by plant extract was found to be a function of their relative concentrations. Inhibitory concentration (IC50) for the plant extract was found to be 0.16 mg/mL. CONCLUSIONS: The present study recorded significant antifilarial effect of Vitex negundo plant extract and contributed toward the development of database for novel drug candidates for lymphatic filariasis.


Subject(s)
Filaricides/pharmacology , Plant Extracts/pharmacology , Plant Leaves/chemistry , Setaria Nematode/drug effects , Vitex/chemistry , Acetates , Animals , Biological Assay , Filaricides/chemistry , Filaricides/isolation & purification , Parasitic Sensitivity Tests , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Setaria Nematode/physiology
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