Antifilarial Activity Research Articles

New Frontier and the Advanced Herbal Approaches for the Treatment of Lymphatic Filariasis

Lymphatic filariasis causes serious health problems in tropical and subtropical developing countries of the world and more than 1.3 billion people are infected. Lymphatic filariasis is disease targeted for elimination by global programme. Global elimination programme have two main objectives one is drug transmission of the parasites and another to provide care for those with the disease. Mass drug administration plays a major for the transmission interruption of disease. Modern synthetic drugs found to be very effective for that purpose, but cause lots of side effects. A large no. of medicinal plants have been claimed to have good antifilarial activity and less side effects. The present review summarizes some present treatment strategies and preliminary studies on herbal approaches of filariasis treatment, which can be investigated further to search of novel herbal drugs to treat filariasis.

Clinical and therapeutic benefits of Centella asiatica

Centella asiatica belongs to family Apiaceae is a traditionally important plant with wide range of therapeutic potential. Plant is traditionally used to treat a broad range of diseases such as diarrhea, hepatitis, measles, toothache, syphilis, leucorrhoea etc. Madecassic acid, asiatic acid, α-terpinene, α-copaene, β-caryophyllene are some of the important bioactive compounds responsible for its antioxidant, antimicrobial, antiulcer, antifilarial, antiviral and various other activities. Present review provides up to date information related to conventional uses, bioactivities and clinical benefits of Centella asiatica. Keywords: Centella asiatica, chemical constituents, ethnomedicinal uses, clinical uses http://dx.doi.org/10.19045/bspab.2014.34004

In vitro, in silico and in vivo studies of ursolic acid as an anti-filarial agent.

As part of our drug discovery program for anti-filarial agents from Indian medicinal plants, leaves of Eucalyptus tereticornis were chemically investigated, which resulted in the isolation and characterization of an anti-filarial agent, ursolic acid (UA) as a major constituent. Antifilarial activity of UA against the human lymphatic filarial parasite Brugia malayi using in vitro and in vivo assays, and in silico docking search on glutathione-s-transferase (GST) parasitic enzyme were carried out. The UA was lethal to microfilariae (mf; LC100: 50; IC50: 8.84 µM) and female adult worms (LC100: 100; IC50: 35.36 µM) as observed by motility assay; it exerted 86% inhibition in MTT reduction potential of the adult parasites. The selectivity index (SI) of UA for the parasites was found safe. This was supported by the molecular docking studies, which showed adequate docking (LibDock) scores for UA (−8.6) with respect to the standard antifilarial drugs, ivermectin (IVM −8.4) and diethylcarbamazine (DEC-C −4.6) on glutathione-s-transferase enzyme. Further, in silico pharmacokinetic and drug-likeness studies showed that UA possesses drug-like properties. Furthermore, UA was evaluated in vivo in B. malayi-M. coucha model (natural infection), which showed 54% macrofilaricidal activity, 56% female worm sterility and almost unchanged microfilaraemia maintained throughout observation period with no adverse effect on the host. Thus, in conclusion in vitro, in silico and in vivo results indicate that UA is a promising, inexpensive, widely available natural lead, which can be designed and developed into a macrofilaricidal drug. To the best of our knowledge this is the first ever report on the anti-filarial potential of UA from E. tereticornis, which is in full agreement with the Thomson Reuter's ‘Metadrug’ tool screening predictions.

In vitro antifilarial activity of Azadirachta indica aqueous extract through reactive oxygen species enhancement.

To evaluate an aqueous preparation from the Azadirachta indica leaves (AEA) against Setaria cervi (S. cervi), a model filarial parasite. In vitro efficacy of AEA was evaluated against S. cervi through estimation of relative motility value, dye exclusion test and MTT assay. Visible morphological alterations were monitored using conventional microscopic techniques in microfilariae and haematoxylin-eosin stained sections of AEA-treated adults. Enhancement of reactive oxygen species in S. cervi treated with AEA was established through alteration in the activity of glutathione S-transferase, superoxide dismutase, catalase, peroxidase and level of superoxide anion and reduced glutathione. In vitro filaricidal activity of AEA is possibly through disturbing redox homeostasis by down-regulating and altering the level of some key antioxidants and regulatory enzymes like reduced glutathione, glutathione S-transferase, superoxide dismutase, catalase and glutathione peroxidase of S. cervi.

IN VITRO SCREENING OF LANNEA COROMANDELICA FOR ANTIFILARIAL ACTIVITY ON CATTLE FILARIAL PARASITE SETARIA CERVI

The current study was aimed to investigate the antifilarial activity of aqueous and ethyl acetate extracts of the leaves of Lannea coromandelica. Both extracts were screened for their total flavonoid content chromatographically. The total amount of flavonoid present in ethyl acetate is much greater than in aqueous extract. Aqueous extract produced initial stimulation effect in whole worm followed by reversible paralysis, whereas on the nerve muscle preparation, the effect was depressant. In the same manner ethyl acetate extract produces irreversible paralysis. The concentrations required to inhibit the movement of the whole worm and nerve muscle preparation for aqueous extract of leaves of L. coromandelica were 300 and 60 µg/mL, respectively, whereas ethyl acetate extract caused inhibition of the whole worm and nerve muscle preparation at 160 and 15 µg/mL, respectively. LC50 and LC90 were 21 and 30 ng/mL, respectively for aqueous, which were 16 and 24 ng/mL for ethyl acetate extracts.

Anti-Onchocerca activity and phytochemical analysis of an essential oil from Cyperus articulatus L.

BackgroundThe lack of a safe and effective adult worm drug and the emergence of resistant animal parasite strains to the only recommended drug, the microfilaricide, ivermectin put many at risk of the devastating effects of the onchocerciasis. The present study was undertaken to investigate the acclaimed anti-Onchocerca activity of the roots/rhizomes of Cyperus articulatus in the traditional treatment of onchocerciasis in North Western Cameroon and to assess the plant as a new source of potential filaricidal lead compounds.MethodsCrude extracts were prepared from the dried plant parts using hexane, methylene chloride and methanol. The antifilarial activity was evaluated in vitro on microfilariae (Mfs) and adult worms of the bovine derived Onchocerca ochengi, a close relative of Onchocerca volvulus. The viabilities of microfilariae and adult male worms were determined based on motility reduction, while for the adult female worms the viability was based on the standard MTT/formazan assay. Cytotoxicity of the active extract was assessed on monkey kidney epithelial cells in vitro and the selectivity indices (SI) were determined. Acute toxicity of the promising extract was investigated in mice. Chemical composition of the active extract was unraveled by GC/MS analysis.ResultsOnly the hexane extract, an essential oil exhibited anti-Onchocerca activity. The oil killed both the microfilariae and adult worms of O. ochengi in a dose manner dependently, with IC50s of 23.4 μg/ml on the Mfs, 23.4 μg/ml on adult male worms and 31.25 μg/ml on the adult female worms. Selectivity indices were 4, 4, and 2.99 for Mfs, adult males and adult females, respectively. At a single limit dose of 2000 mg/kg body weight, none of 6 mice that received the essential oil by gavage died. GC/MS analysis revealed the presence of terpenoids, hydrocarbons and fatty acids or fatty acid derivatives as components of the oil.ConclusionsThe essential oil from the roots/rhizomes of Cyperus articulatus is active against O. ochengi microfilariae and adult worms in vitro in a dose dependent manner, hence may provide a source of new anti-filarial compounds. The results also support the traditional use of C. articulatus in the treatment of human onchocerciasis.

Antifilarial effect of ursolic acid from Nyctanthes arbortristis: Molecular and biochemical evidences

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.

Anti-onchocerca Metabolites from Cyperus articulatus: Isolation, In Vitro Activity and In Silico 'Drug-Likeness'.

The aims of this investigation were to isolate active ingredients from the roots/rhizomes of Cyperus articulatus used as herbal medicine in Cameroon for the treatment of human onchocerciasis and to assess the efficacy of the metabolites on the Onchocerca worm. The antifilarial activity was evaluated in vitro on microfilariae (Mfs) and adult worms of the bovine derived Onchocerca ochengi, a close relative of Onchocerca volvulus. Cytotoxicity was assessed in vitro on monkey kidney epithelial cells. The structures of the active compounds were determined using spectroscopic methods and their drug-likeness evaluated using Lipinski parameters. Two secondary metabolites, AMJ1 [containing mustakone (1) as the major component] and linoleic acid or (9Z,12Z)-octadeca-9,12-dienoic acid (2) were isolated. Both compounds were found to kill both the microfilariae and adult worms of O. ochengi in a dose dependent manner. The IC50s for AMJ1 were 15.7 µg/mL for Mfs, 17.4 µg/mL for adult males and 21.9 µg/mL for adult female worms while for linoleic acid the values were, 15.7 µg/mL for Mfs, 31.0 µg/mL for adult males and 44.2 µg/mL for adult females. The present report provides the first ever evidence of the anti-Onchocerca efficacy of AMJ1 and linoleic acid. Thus, these secondary metabolites may provide a lead for design and development of new antifilarial agents.Electronic supplementary materialThe online version of this article (doi:10.1007/s13659-014-0023-5) contains supplementary material, which is available to authorized users.

Synthesis and antifilarial activity of chalcone–thiazole derivatives against a human lymphatic filarial parasite, Brugia malayi

Here we report the synthesis of novel chalcone–thiazole compounds and their antifilarial activity. The antifilarial properties of these hybrids were assessed against microfilariae as well as adult worms of Brugia malayi. Among all the synthesized compounds, only two compounds, namely 4g and 4n were identified to be promising in vitro. These active compounds were tested in B. malayi-jird (Meriones unguiculatus) and B. malayi–Mastomys coucha models. Compound 4n showed 100% embryostatic effect and 49% macrofilaricidal in jirds and M. coucha models, respectively. This study provides a new structural clue for the development of novel antifilarial lead molecules.

Antifilarial activity of Butea monosperma L. leaves extracts against Setaria cervi

Abstract Background: Usage of herbal drugs in traditional medicine is quite well known but largely empirical. Hence the present study was designed to screen the in vitro antifilarial effect of Butea

Therapeutic efficacy of poly (lactic-co-glycolic acid) nanoparticles encapsulated ivermectin (nano-ivermectin) against brugian filariasis in experimental rodent model

The present study reports on the antifilarial activity of poly (lactic-co-glycolic acid) nanoparticles encapsulated ivermectin (nano-IVM) against human lymphatic filariid Brugia malayi in rodent host Mastomys coucha. Nano-IVM was prepared and optimized by nanoprecipitation method. The selected nano-IVM (F5) showed a uniform spherical shape with 96 nm diameter and 74.12 % entrapment efficiency, and when used at a suboptimal dose of 100 μg/kg body weight, completely eliminated filarial parasites from systemic circulation on 60 days post-infection in animals inflicted with B. malayi. In contrast, the coadministration of nano-IVM (F5) along with standard filaricide diethylcarbamazine (DEC) was found to be competent enough to suppress microfilarial stage of parasites and successfully eliminated microfilaria at 45 days posttreatment. However, the free form of both the drugs alone or in combination was unable to impart such suppression and followed by recurrence of the infection. Interestingly, nano-IVM (F5) was also found to be effective against adult stage parasites causing 36.67 % worm mortality and 75.89 % in combination with DEC; however, female sterilization remain almost similar. Thus, the combination of entrapped IVM with DEC exhibited enhanced microfilaricidal and marginally better macrofilaricidal efficacy than any of the single formulation or drugs combination.

Antifilarial effects of polyphenol rich ethanolic extract from the leaves of Azadirachta indica through molecular and biochemical approaches describing reactive oxygen species (ROS) mediated apoptosis of Setaria cervi

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.

New chemotypes for wALADin1-like inhibitors of delta-aminolevulinic acid dehydratase from Wolbachia endobacteria

Substituted benzimidazoles of the wALADin1-family have recently been identified as a new class of species-selective inhibitors of delta-aminolevulinic acid dehydratase (ALAD) from Wolbachia endobacteria of parasitic filarial worms. Due to its Wolbachia-dependent antifilarial activity, wALADin1 is a starting point for the development of new drugs against filarial nematodes. We now present several other chemotypes of ALAD inhibitors that have been identified based upon their molecular similarity to wALADin1. A tricyclic quinoline derivative (wALADin2) with a different inhibitory mechanism and improved inhibitory potency and selectivity may represent an improved drug lead candidate.

Diarylheptanoid compounds from Alnus nepalensis express in vitro and in vivo antifilarial activity

A large number of medicinal plants remain to be explored for antifilarial compounds. In the present study a crude methanolic extract of leaves of Alnus nepalensis, chloroform- and n-butanol-partitioned fractions from the crude extract and 6 bioactivity-guided isolated compounds including two new diarylheptanoid from the fractions were assayed for microfilaricidal, macrofilaricidal and female worm sterilizing activity using the lymphatic filariid Brugia malayi in in vitro and in vivo systems. In vitro, the crude methanolic extract exerted better microfilaricidal (LC100: 15.63μg/ml, IC50: 6.00μg/ml) than macrofilaricidal (LC100: >250; IC50: 88μg/ml) activity whereas chloroform and n-butanol fractions were more macrofilaricidal (LC100: 125 and 31.25μg/ml; IC50: 13.14 and 11.84, respectively) than microfilaricidal (LC100: 250–500μg/ml, IC50: 44.16μg/ml). In addition, n-butanol fraction also caused 74% inhibition in MTT reduction potential of the adult worms. In vivo (doses: crude: 100–200mg/kg; fractions: 100mg/kg, i.p.×5 days) the chloroform fraction exerted >50% macrofilaricidal activity whereas methanolic extract and n-butanol fraction produced 38–40% macrofilaricidal action along with some female sterilizing efficacy. Of the 5 diarylheptanoid compounds isolated, alnus dimer, and (5S)-5-hydroxy-1-(4-hydroxyphenyl)-7-(3,4-dihydroxyphenyl)-3-heptanone were found to show the most potent with both macrofilaricidal (LC100: 15.63μg/ml, IC50: 6.57–10.31μg/ml) and microfilaricidal (LC100: 31.25–62.5μg/ml, IC50: 11.05–22.10μg/ml) activity in vitro. These findings indicate that the active diarylheptanoid compounds may provide valuable lead for design and development of new antifilarial agent(s).

Improved antifilarial activity of ivermectin in chitosan–alginate nanoparticles against human lymphatic filarial parasite, Brugia malayi

The current antifilarial treatments are not up to the mark partly due to deep location of filarial parasites in the human lymphatic system. We report here on the improvement in the antifilarial activity of ivermectin (IVM) using chitosan-alginate nanoparticles prepared by modified complex coacervation method. The nanoparticles were spherical having 155 nm size and 4.56 and 75.67% loading and entrapment efficiency respectively for IVM. The delivery system maintained the sustained release and significantly augmented the microfilaricidal (MIF) activity at a single low dose (200 μg/kg body weight, subcutaneously) in contrast to much higher dose of free ivermectin (400 μg/kg body weight, subcutaneously) against human lymphatic filariid, Brugia malayi in rodent host, Mastomys coucha. To substantiate increase in MIF activity, pharmacokinetics study was designed on Wistar rats which revealed a greater peak plasma concentration (45.3 ± 1.79 ng/ml), area under the concentration curve (298 ± 38.7 ng d/ml) and extended mean residence time (23.4 ± 8.56 days)of IVM in chitosan-alginate nanoparticles. Administration of 25 mg/kg of diethylcarbamazine following nanoparticle therapy significantly improved the MIF and macrofilaricidal action of encapsulated drug and was considered superior in this study.

Antifilarial activity of ethyl acetate extract of Vitex negundo leaves in vitro

ObjectiveTo evaluate the possible antifilarial effect of ethyl acetate extract of Vitex negundo (Verbenaceae) leaves against Setaria cervi filarial parasite in vitro. MethodsIn 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. ResultsIn 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. ConclusionsThe 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.

Molecular Characterization of an rsmD -Like rRNA Methyltransferase from the Wolbachia Endosymbiont of Brugia malayi and Antifilarial Activity of Specific Inhibitors of the Enzyme

The endosymbiotic organism Wolbachia is an attractive antifilarial drug target. Here we report on the cloning and expression of an rsmD-like rRNA methyltransferase from the Wolbachia endosymbiont of Brugia malayi, its molecular properties, and assays for specific inhibitors. The gene was found to be expressed in all the major life stages of B. malayi. The purified enzyme expressed in Escherichia coli was found to be in monomer form in its native state. The activities of the specific inhibitors (heteroaryl compounds) against the enzyme were tested with B. malayi adult and microfilariae for 7 days in vitro at various concentrations, and NSC-659390 proved to be the most potent compound (50% inhibitory concentration [IC50], 0.32 μM), followed by NSC-658343 (IC50, 4.13 μM) and NSC-657589 (IC50, 7.5 μM). On intraperitoneal administration at 5 mg/kg of body weight for 7 days to adult jirds into which B. malayi had been transplanted intraperitoneally, all the compounds killed a significant proportion of the implanted worms. A very similar result was observed in infected mastomys when inhibitors were administered. Docking studies of enzyme and inhibitors and an in vitro tryptophan quenching experiment were also performed to understand the binding mode and affinity. The specific inhibitors of the enzyme showed a higher affinity for the catalytic site of the enzyme than the nonspecific inhibitors and were found to be potent enough to kill the worm (both adults and microfilariae) in vitro as well as in vivo in a matter of days at micromolar concentrations. The findings suggest that these compounds be evaluated against other pathogens possessing a methyltransferase with a DPPY motif and warrant the design and synthesis of more such inhibitors.

Antifilarial Activity of Constituents of Calophyllum inophyllum and their Derivatives

Elephantiasis is a classic sign of late-stage lymphatic filariasis. Although the infection can be treated with drugs, the chronic conditions may not be curable by currently available antifilarial drugs. In continuation of our drug discovery program, we identified potent antifilarial activity in a calophyllic acid and isocalophllic acid mixture, which was isolated from the leaves of Calophyllum inophyllum. The mixture inhibited the motility of adult Brugia malayi worms and microfilariae stage parasites at a concentration of 15.6 μg/mL with an IC50 of 2.1 and 5.5 μg/mL, respectively, in in vitro studies. The diastereomeric mixture also exhibited moderate in vivo antifilarial activity in the jird ( Meriones unguiculatus) model. A few derivatives of calophyllic acid and isocalophyllic acid were prepared and one of the amide derivatives turned out to be the most promising compound against the adult (MIC: 3.9 and IC50: 0.32 μg/mL) and microfilariae stage (MIC:1.9 and IC50: 0.26 μg/mL) parasites with a high selectivity index. The amide derivative has superior activity than the parent natural product, as well as the standard antifilarial drug, ivermectin.

Synthesis and Macrofilaricidal Activity of Substituted 2‐Hydroxy/5‐Hydroxy/2‐Methyl‐1,4‐Naphthoquinones

Abstract Preclinical Research Lymphatic filariasis is a disfiguring disease caused by parasitic worms that destroy the human lymphatic system leading to substantial morbidity. The current drug of choice for the treatment of filariasis is diethylcarbamazine and ivermectin with albendazole which are only effective against the microfilaria, leaving the adult worm unaffected, requiring the development of “adulticidal drugs.” Thirty amino substituted 2‐hydroxy/5‐hydroxy/2‐methyl‐1,4‐naphthoquinones were synthesized via the reaction of 2‐hydroxy/5‐hydroxy/2‐methyl‐1,4‐naphthoquinones with different primary and secondary amines. Compounds 1–30 were evaluated for in vitro antifilarial activity against the adult bovine filarial worm Setaria digitata as assessed by worm motility and MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) reduction assays. The mutagenecity, tumerogenecity, irritantancy, reproductive toxicity, drug score, druglike, and cLogP properties were calculated using OSIRIS property predictor. Ten compounds showed macrofilaricidal activity with ED50 values ranging between 0.086 and 7.6 μM. Taking into account the biological effects and the promising drug‐like profiles of these compounds, these represent valid leads for the development of antifilarial agents against adult filarial worm.

A Selective Inhibitor of Heme Biosynthesis in Endosymbiotic Bacteria Elicits Antifilarial Activity In Vitro

Lymphatic filariasis and onchocerciasis are severe diseases caused by filarial worms and affect more than 150 million people worldwide. Endosymbiotic α-proteobacteria Wolbachia are essential for these parasites throughout their life cycle. Using a high-throughput chemical screen, we identified a benzimidazole compound, wALADin1, that selectively targets the δ-aminolevulinic acid dehydratase (ALAD) of Wolbachia (wALAD) and exhibits macrofilaricidal effects on Wolbachia-containing filarial worms invitro. wALADin1 is a mixed competitive/noncompetitive inhibitor that interferes with the Mg(2+)-induced activation of wALAD. This mechanism inherently excludes activity against the Zn(2+)-dependent human ortholog and might be translatable to Mg(2+)-responsive orthologs of other bacterial or protozoan pathogens. The specificity profile of wALADin1 derivatives reveals chemical features responsible for inhibitory potency and species selectivity. Our findings validate wALADins as a basis for developing potent leads that meet current requirements for antifilarial drugs.