Filaricidal activity of Daniellia oliveri and Psorospermum febrifugum extracts.

BACKGROUND: Drugs currently used for controlling onchocerciasis and lymphatic filariasis (LF) are mainly microfilaricidal, with minimal or no effect on the adult worms. For efficient management of these diseases, it is necessary to search for new drugs with macrofilaricidal activities that can be used singly or in combination with existing ones. Daniellia oliveri and Psorospermum febrifugum are two plants commonly used in the local management of these infections in Bambui, a township in the North West Region of Cameroon, but there is currently no documented scientific evidence to support their claimed anthelmintic efficacy and safety. The aim of this study was to provide evidence in support of the search for means to eliminate these diseases by screening extracts and chromatographic fractions isolated from these plants for efficacy against the parasitic roundworms Onchocerca ochengi and Brugia pahangi. METHODS: The viability of O. ochengi adult worms was assessed using the MTT/formazan assay. Fully confluent monkey kidney epithelial cells (LLC-MK2) served as the feeder layer for the O. ochengi microfilariae (mfs) assays. Viability of the mfs was assessed by microscopic examination for mean motility scoring (relative to the negative control) every 24 h post addition of an extract. The Worminator system was used to test the effects of the extracts on adult B. pahangi motility, and mean motility units were determined for each worm. Cytotoxicity of the active extracts on N27 cells was assessed using the MTS assay. RESULTS: Extracts from D. oliveri and P. febrifugum were effective against the adult roundworms O. ochengi and B. pahangi. Interestingly, extracts showing macrofilaricidal activities against O. ochengi also showed activity against O. ochengi mfs. The hexane stem bark extract of D. oliveri (DOBHEX) was more selective for adult O. ochengi than for mfs, with a half maximal and 100% inhibitory concentration (IC50 and IC100, respectively) against adult O. ochengi of 13.9 and 31.3 µg/ml, respectively. The in vitro cytotoxicity of all active extracts on N27 cells showed selective toxicity for parasites (selectivity index > 1). Bioassay-guided fractionation of the extracts yielded fractions with activity against adult B. pahangi, thus confirming the presence of bioactive principles in the plant extracts. CONCLUSIONS: Our study supports the use of D. oliveri and P. febrifugum in the traditional treatment of onchocerciasis and LF. The further purification of active extracts from these plants could yield lead compounds for filarial drug discovery and development.

Antischistosomal, antionchocercal and antitrypanosomal potentials of some Ghanaian traditional medicines and their constituents.

BACKGROUND: Ghana is endemic for some neglected tropical diseases (NTDs) including schistosomiasis, onchocerciasis and lymphatic filariasis. The major intervention for these diseases is mass drug administration of a few repeatedly recycled drugs which is a cause for major concern due to reduced efficacy of the drugs and the emergence of drug resistance. Evidently, new treatments are needed urgently. Medicinal plants, on the other hand, have a reputable history as important sources of potent therapeutic agents in the treatment of various diseases among African populations, Ghana inclusively, and provide very useful starting points for the discovery of much-needed new or alternative drugs. METHODOLOGY/PRINCIPAL FINDINGS: In this study, extracts of fifteen traditional medicines used for treating various NTDs in local communities were screened in vitro for efficacy against schistosomiasis, onchocerciasis and African trypanosomiasis. Two extracts, NTD-B4-DCM and NTD-B7-DCM, prepared from traditional medicines used to treat schistosomiasis, displayed the highest activity (IC50 = 30.5 µg/mL and 30.8 µg/mL, respectively) against Schistosoma mansoni adult worms. NTD-B2-DCM, also obtained from an antischistosomal remedy, was the most active against female and male adult Onchocera ochengi worms (IC50 = 76.2 µg/mL and 76.7 µg/mL, respectively). Antitrypanosomal assay of the extracts against Trypanosoma brucei brucei gave the most promising results (IC50 = 5.63 µg/mL to 18.71 µg/mL). Incidentally, NTD-B4-DCM and NTD-B2-DCM, also exhibited the greatest antitrypanosomal activities (IC50 = 5.63 µg/mL and 7.12 µg/mL, respectively). Following the favourable outcome of the antitrypanosomal screening, this assay was selected for bioactivity-guided fractionation. NTD-B4-DCM, the most active extract, was fractionated and subsequent isolation of bioactive constituents led to an eupatoriochromene-rich oil (42.6%) which was 1.3-fold (IC50 <0.0977 µg/mL) more active than the standard antitrypanosomal drug, diminazene aceturate (IC50 = 0.13 µg/mL). CONCLUSION/SIGNIFICANCE: These findings justify the use of traditional medicines and demonstrate their prospects towards NTDs drug discovery.

Modeling the metabolic interplay between a parasitic worm and its bacterial endosymbiont allows the identification of novel drug targets.

The filarial nematode Brugia malayi represents a leading cause of disability in the developing world, causing lymphatic filariasis in nearly 40 million people. Currently available drugs are not well-suited to mass drug administration efforts, so new treatments are urgently required. One potential vulnerability is the endosymbiotic bacteria Wolbachia-present in many filariae-which is vital to the worm. Genome scale metabolic networks have been used to study prokaryotes and protists and have proven valuable in identifying therapeutic targets, but have only been applied to multicellular eukaryotic organisms more recently. Here, we present iDC625, the first compartmentalized metabolic model of a parasitic worm. We used this model to show how metabolic pathway usage allows the worm to adapt to different environments, and predict a set of 102 reactions essential to the survival of B. malayi. We validated three of those reactions with drug tests and demonstrated novel antifilarial properties for all three compounds.

An Insight into the Discovery of Potent Antifilarial Leads Against Lymphatic Filariasis.

BACKGROUND AND OBJECTIVES: Lymphatic filariasis is a neglected tropical disease caused by infection with filarial worms that are transmitted through mosquito bites. Globally, 120 million people are infected, with nearly 40 million people disfigured and disabled by complications such as severe swelling of the legs (elephantiasis) or scrotum (hydrocele). Current treatments (ivermectin, diethylcarbamazine) have limited effects on adult parasites and produce side effects; therefore, there is an urgent to search for new antifilarial agents. Numerous studies on the antifilarial activity of pure molecules have been reported accross the recent literature. The present study describes the current standings of potent antifilarial compounds against lymphatic filariasis. METHODS: A literature search was conducted for naturally occurring and synthetic antifilarial compounds by referencing textbooks and scientific databases (SciFinder, PubMed, Science Direct, Wiley, ACS, SciELO, Google Scholar, and Springer, among others) from their inception until September 2019. RESULTS: Numerous compounds have been reported to exhibit antifilarial acitivity in adult and microfilariae forms of the parasites responsible for lymphatic filariasis. In silico studies of active antifilarial compounds (ligands) showed molecular interactions over the protein targets (trehalose-6-phosphate phosphatase, thymidylate synthase, among others) of lymphatic filariasis, and supported the in vitro results. CONCLUSION: With reference to in vitro antifilarial studies, there is evidence that natural and synthetic products can serve as basic scaffolds for the development of antifilarial agents. The optimization of the most potent antifilarial compounds can be further performed, followed by their in vivo studies.

In Vitro and In Vivo Antifilarial Activity of Standardized Extract of Calotropis procera Flowers against Brugia malayi.

BACKGROUND: Lymphatic filariasis (LF) is a parasitic disease that causes permanent disability (elephantiasis). Currently used antifilarial drugs are failing to control LF and there is resurgence in some areas. Looking for new antifilarial leads, we found that Calotropis procera plant parts have been used in traditional medicine for alleviating elephantiasis but the antifilarial activity is not known. OBJECTIVE: In the present study, the antifilarial activity of ethanolic extract (A001) and its hexane fraction (F001) of C. procera flowers was investigated using the human filarial parasite Brugia malayi. METHODS: A001 and F001 were tested for antifilarial activity using motility and 3-(4,5-dimethylthiazol-2- yl)-2,5 diphenyltetrazolium bromide (MTT) assays (in vitro) and in the rodent models B. malayi- Meriones unguiculatus and B. malayi-Mastomys coucha. In the rodent models, A001 and F001 were administered orally for 5 consecutive days, and the adult worm burden and course of microfilaraemia were determined. RESULTS: Both A001 and F001 showed microfilaricidal and macrofilaricidal activity in vitro. In animal models, A001 killed ~49-54% adult worms. In M. coucha model, F001 killed 12-60% adult worms in a dose (125-500 mg/kg) dependent manner; A001 and F001 suppressed microfilaraemia till days 91 and 35 post initiation of treatment, respectively. HPTLC revealed 0.61% lupeol, 0.50% ß-sitosterol and 1.50% triacontanol in F001. CONCLUSION: Flowers of C. procera have definite microfilaricidal and macrofilaricidal activities. Whether this activity is due to lupeol, ß-sitosterol and triacontanol found in the hexane fraction remains to be investigated. This is the first report on the antifilarial efficacy of flowers of the plant C. procera.

Macro and microfilaricidal activities of extracts of Annona senegalensis and Milletia comosa against Onchocerca ochengi and Loa loa.

Despite the efforts employed for the control of onchocerciasis, the latter has remained a significant public health problem, due mainly to the lack of safe and effective adult worm drugs and/or microfilaricides that do not kill Loa loa microfilariae (mf). Serious adverse events have been encountered after administering ivermectin to some onchocerciasis patients coinfected with Loa loa. There is therefore, an urgent need for a macro and/or microfilaricidal drug which kills Onchocerca but not L. loa microfilariae. A total of 12 crude extracts from Milletia comosa and Annona senegalensis were prepared and screened in vitro against the bovine species of Onchocerca, O. ochengi, and L. loa mf from humans. Mf and male worm viabilities were determined by motility scoring using microscopy at 120 h of incubation with drug, while adult female worm viability and cytotoxicity were determined biochemically by MTT/formazan colorimetry after 120 h of incubation with drug. Out of the 12 extracts, all 6 from M. comosa and 4 from A. senegalensis were active against male, female and mf of O. ochengi. The hexane extract from M. comosa leaves (MCL hex) was the most active with IC50 values of 1.38, 0.86 and 17.74 µg/mL for O. ochengi adult males, adult female and the mf, respectively. About 58% of the extracts were more active against O. ochengi than L. loa mf. These results demonstrate that these extracts contain active principles that kill Onchocerca parasite and to a lesser extent L. loa, and suggest that they can be fractionated for isolation of lead molecules for the safe treatment of onchocerciasis.

Development of a preliminary in vitro drug screening assay based on a newly established culturing system for pre-adult fifth-stage Onchocerca volvulus worms.

BACKGROUND: The human filarial parasite Onchocerca volvulus is the causative agent of onchocerciasis (river blindness). It causes blindness in 270,000 individuals with an additional 6.5 million suffering from severe skin pathologies. Current international control programs focus on the reduction of microfilaridermia by annually administering ivermectin for more than 20 years with the ultimate goal of blocking of transmission. The adult worms of O. volvulus can live within nodules for over 15 years and actively release microfilariae for the majority of their lifespan. Therefore, protracted treatment courses of ivermectin are required to block transmission and eventually eliminate the disease. To shorten the time to elimination of this disease, drugs that successfully target macrofilariae (adult parasites) are needed. Unfortunately, there is no small animal model for the infection that could be used for discovery and screening of drugs against adult O. volvulus parasites. Here, we present an in vitro culturing system that supports the growth and development of O. volvulus young adult worms from the third-stage (L3) infective stage. METHODOLOGY/PRINCIPAL FINDINGS: In this study we optimized the culturing system by testing several monolayer cell lines to support worm growth and development. We have shown that the optimized culturing system allows for the growth of the L3 worms to L5 and that the L5 mature into young adult worms. Moreover, these young O. volvulus worms were used in preliminary assays to test putative macrofilaricidal drugs and FDA-approved repurposed drugs. CONCLUSION: The culture system we have established for O. volvulus young adult worms offers a promising new platform to advance drug discovery against the human filarial parasite, O. volvulus and thus supports the continuous pursuit for effective macrofilaricidal drugs. However, this in vitro culturing system will have to be further validated for reproducibility before it can be rolled out as a drug screen for decision making in macrofilaricide drug development programs.

Polyphenol enriched ethanolic extract of Cajanus scarabaeoides (L.) Thouars exerts potential antifilarial activity by inducing oxidative stress and programmed cell death.

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.

Assessment of macrofilaricidal activity of leaf extracts of Terminalia sp. against bovine filarial parasite Setaria cervi.

Antifilarial potential of three medicinal plants namely, Terminalia bellerica, Terminalia chebula and Terminalia catappa was explored using Setaria cervi, a bovine filarial parasite at concentrations of 2.5, 5 and 10mg/ml. Amongst all the extracts, methanol extract of T. bellerica showed highest macrofilaricidal activity i.e. 84.63±1.11 at 10mg/ml in MTT reduction assay with IC50 value of 2.7mg/ml. which was better than the standard DEC i.e. 79.22±3.1% at 10mg/ml with IC50 value 2.84mg/ml. Other plant extracts showed mild in vitro macrofilaricidal activity. T. bellerica methanol extract exhibited significant GST activity of 18.86±0.21 and 12.83±0.03µM/ml/min at 5 and 10mg/ml with percentage inhibition value of 73.96% and 82.29% respectively. DEC showed GST activity value of 40.03±4.14 and 21.48±6.44µM/ml/min with percentage inhibition value of 21.76% and 58.01% at 5 and 10mg/ml respectively. Thus, methanol extract of leaves of T. bellerica exhibited highly significant antifilarial potential and needs detailed analysis.

Filaricidal activities on Onchocerca ochengi and Loa loa, toxicity and phytochemical screening of extracts of Tragia benthami and Piper umbellatum.

BACKGROUND: Onchocerciasis is the world's second leading infectious cause of blindness. Its control is currently hampered by the lack of a macrofilaricidal drug and by severe adverse events observed when the lone recommended microfilaricide, ivermectin is administered to individuals co-infected with Loa loa. Therefore, there is the need for a safe and effective macrofilaricidal drug that will be able to cure the infection and break transmission cycles, or at least, an alternative microfilaricide that does not kill L. loa microfilariae (mf). METHODS: Fourteen extracts from two medicinal plants, Tragia benthami and Piper umbellatum were screened in vitro against Onchocerca ochengi parasite and L. loa mf. Activities of extracts on male worms and microfilariae were assessed by motility reduction, while MTT/Formazan assay was used to assess biochemically the death of female worms. Cytotoxicity and acute toxicity of active extracts were tested on monkey kidney cells and Balb/c mice, respectively. RESULTS: At 500 µg/mL, all extracts showed 100 % activity on Onchocerca ochengi males and microfilariae, while 9 showed 100 % activity on female worms. The methylene chloride extract of Piper umbellatum leaves was the most active on adult male and female worms (IC50s: 16.63 µg/mL and 35.65 µg/mL, respectively). The three most active extracts on Onchocerca ochengi females were also highly active on Loa loa microfilariae, with IC50s of 35.12 - 13.9 µg/mL. Active extracts were generally more toxic to the worms than to cells and showed no acute toxicity to Balb/c mice. Phytochemical screening revealed the presence of saponins, steroids, tannins and flavanoids in the promising extracts. CONCLUSIONS: These results unfold potential sources of novel anti-Onchocerca lead compounds and validate the traditional use of the plants in onchocerciasis treatment.

Antifilarial and Antibiotic Activities of Methanolic Extracts of Melaleuca cajuputi Flowers.

We evaluated the activity of methanolic extracts of Melaleuca cajuputi flowers against the filarial worm Brugia pahangi and its bacterial endosymbiont Wolbachia. Anti-Wolbachia activity was measured in worms and in Aedes albopictus Aa23 cells by PCR, electron microscopy, and other biological assays. In particular, microfilarial release, worm motility, and viability were determined. M. cajuputi flower extracts were found to significantly reduce Wolbachia endosymbionts in Aa23 cells, Wolbachia surface protein, and microfilarial release, as well as the viability and motility of adult worms. Anti-Wolbachia activity was further confirmed by observation of degraded and phagocytized Wolbachia in worms treated with the flower extracts. The data provided in vitro and in vivo evidence that M. cajuputi flower extracts inhibit Wolbachia, an activity that may be exploited as an alternative strategy to treat human lymphatic filariasis.

Antifilarial activity of diterpenoids from Taxodium distichum.

BACKGROUND: Lymphatic filariasis caused by Wuchereria bancrofti, Brugia malayi and B. timori, is a debilitating disease with an adverse social and economic impact. The infection remains unabated in spite of treatment with existing antifilarial drugs diethylcarbamazine (DEC) and ivermectin which are chiefly microfilaricides. There is therefore, need for macrofilaricides, embryostatic agents and better microfilaricides. In the present study we explored the antifilarial potential of crude extract and its molecular fractions of the plant Taxodium distichum using in vitro assay systems and rodent models of B. malayi infection. METHODS: Ethanolic extract (A001) of aerial parts of T. distichum was solvent fractionated and sub-fractionated. Four molecules, 3-Acetoxylabda-8(20), 13-diene-15-oic acid (K001), Beta-sitosterol (K002), labda-8(20),13-diene-15-oic acid (K003) and Metasequoic acid A (K004) were isolated from the fractions and their structure determined by spectroscopic analysis. The extract, subfractions and molecules were evaluated for antifilarial activity against B. malayi by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) reduction and motility assays in vitro and in two animal models, Meriones unguiculatus and Mastomys coucha, harbouring B. malayi infection. RESULTS: A001 was effective in killing microfilariae (mf) and adult worms in vitro. The diterpenoid K003 produced 100 % reduction in motility of both mf and adult worms and > 80 % inhibition in MTT reduction potential of adult female worms. In B. malayi-M. unguiculatus model, A001 killed all the adult worms in > 80 % of infected animals. K003 was embryostatic (> 95 %) in this model. In the B. malayi-M. coucha model, K003 killed ~54 % of adult worms (macrofilaricidal activity) and rendered > 36 % female worms sterile; it also stopped any further rise in microfilaraemia after day 42 post-initiation of treatment. CONCLUSION: Ethanolic extract of aerial parts of the plant T. distichum possesses potent antifilarial activity and the active principle was localised to K003 which showed significant macrofilaricidal activity and late suppression of peripheral microfilaraemia and some embryostatic activity. These findings indicate that labdane diterpenoid molecule(s) may provide valuable leads for design and development of new macrofilaricidal agent(s). To the best of our knowledge, this is the first report on antifilarial efficacy of products from the plant T. distichum.

Synthesis and biological evaluation of 4-oxycoumarin derivatives as a new class of antifilarial agents.

A series of 4-oxycoumarin derivatives was synthesized, characterized and evaluated in vitro and in vivo for antifilarial activity against the human lymphatic filarial parasite, Brugia malayi. A majority of the compounds studied showed potent in vitro activity with low IC50 values in the micro molar (µM) range (0.014-1.73 and 0.0056-0.43) against adult worms and microfilariae, respectively. Compounds 8 and 9 were identified to be the most promising antifilarial candidate molecules exhibiting activity in the nanomolar (nM) range. The IC50 values for compound 8 were 14 nM and 5.6 nM while for compound 9 were 94 nM and 13 nM, respectively, for adult worm and microfilaria. These two compounds also displayed promising adulticidal activity (74.9 ± 4.8% and 69.4 ± 2.8%, respectively) in the primary rodent (jird) screen. This study also serves as a starting point for investigating structure-activity relationship with different amino substituents.

Diospyros perigrena bark extract induced apoptosis in filarial parasite Setaria cervi through generation of reactive oxygen species.

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.

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.

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

BACKGROUND: The 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. METHODS: Crude 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. RESULTS: Only 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. CONCLUSIONS: The 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.

Virtual screening of traditional Chinese medicine (TCM) database: identification of fragment-like lead molecules for filariasis target asparaginyl-tRNA synthetase.

Lymphatic filariasis (LF) is a vector borne infectious disease caused by the nematode Wuchereria bancrofti, Brugia malayi, and Brugia timori. Over 120 million people are affected by LF in the world, of which two-thirds are in Asia. The infection restricts the normal flow of lymph from the infected area resulting in swelling of the extremities and causing permanent disability. As the available drugs for the treatment of LF are becoming ineffective due to the development of resistance, there is an urgent need to find new leads for drug development. In this study, asparaginyl-tRNA synthetase (AsnRS; PDB ID: 2XGT) essential for the protein bio-synthesis in the filarial nematode was used to carry out virtual screening (VS) of plant constituents from traditional Chinese medicine (TCM) database. Docking as well as E-pharmacophore based VS were carried out to identify the hits. The top scoring hits, Agri 1 (1,3,8-trihydroxy-4,5-dimethoxyxanthen-9-one-3-O-beta-D-glucopyranoside) and Agri 2 (5,7-dihydroxy-2-propylchromone 7-O-beta-D-glucopyranoside), constituents of Agrimonia pilosa, were selected for molecular dynamics (MD) simulation study for 10 ns. MD simulation showed that both the glycosides Agri 1 and Agri 2 were forming stable interactions with the target protein. Moreover, docking and MD simulation of the lead A (1,3,8-trihydroxy-4,5-dimethoxyxanthen-9-one; Mol. Wt.: 304.25; CLogP: 3.07) and lead B (5,7-dihydroxy-2-propylchromone; Mol. Wt.: 220.22; CLogP: 3.02), the aglycones of Agri 1 and Agri 2, respectively, were carried out with the target AsnRS. The in silico investigations of the aglycones suggest that the lead B could be a suitable fragment-like lead molecule for anti-filarial drug discovery.

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.

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).