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.

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.

Evaluation of in vitro culture systems for the maintenance of microfilariae and infective larvae of Loa loa.

BACKGROUND: Suitable and scalable in vitro culture conditions for parasite maintenance are needed to foster drug research for loiasis, one of the neglected tropical diseases which has attracted only limited attention over recent years, despite having important public health impacts. The present work aims to develop adequate in vitro culture systems for drug screening against both microfilariae (mf) and infective third-stage larvae (L3) of Loa loa. METHODS: In vitro culture conditions were evaluated by varying three basic culture media: Roswell Park Memorial Institute (RPMI-1640), Dulbecco's modified Eagle's medium (DMEM) and Iscove's modified Dulbecco's medium (IMDM); four sera/proteins: newborn calf serum (NCS), foetal bovine serum (FBS), bovine serum albumin (BSA) and the lipid-enriched BSA (AlbuMax® II, ALB); and co-culture with the Monkey Kidney Epithelial Cell line (LLC-MK2) as a feeder layer. The various culture systems were tested on both mf and L3, using survival (% motile), motility (T90 = mean duration (days) at which at least 90% of parasites were fully active) and moulting rates of L3 as the major criteria. The general linear model regression analysis was performed to assess the contribution of each variable on the viability of Loa loa L3 and microfilarie. All statistical tests were performed at 95% confidence interval. RESULTS: Of the three different media tested, DMEM and IMDM were the most suitable sustaining the maintenance of both L. loa L3 and mf. IMDM alone could sustain L3 for more than 5 days (T90 = 6.5 ± 1.1 day). Serum supplements and LLC-MK2 co-cultures significantly improved the survival of parasites in DMEM and IMDM. In co-cultures with LLC-MK2 cells, L. loa mf were maintained in each of the three basic media (T90 of 16.4-19.5 days) without any serum supplement. The most effective culture systems promoting significant moulting rate of L3 into L4 (at least 25%) with substantial maintenance time were: DMEM + BSA, DMEM + NCS, DMEM-AlbuMax®II, DMEM + FBS all in co-culture with LLC-MK2, and IMDM + BSA (1.5%), DMEM + FBS (10%) and DMEM + NCS (5%) without feeder cells. DMEM + 1% BSA in co-culture scored the highest moulting rate of 57 of 81 (70.37%). The factors that promoted L. loa mf viability included feeder cells (ß = 0.490), both IMDM (ß = 0.256) and DMEM (ß = 0.198) media and the protein supplements NCS (ß = 0.052) and FBS (ß = 0.022); while for L. loa L3, in addition to feeder cells (ß = 0.259) and both IMDM (ß = 0.401) and DMEM (ß = 0.385) media, the protein supplements BSA (ß = 0.029) were found important in maintaining the worm motility. CONCLUSIONS: The findings from this work display a range of culture requirements for the maintenance of Loa loa stages, which are suitable for developing an effective platform for drug screening.

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.

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.

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).

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

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.

Glycyrrhetinic acid and its analogs: a new class of antifilarial agents.

Although a number of chemicals have been isolated from Glycyrrhiza glabra, only a few have been evaluated for their biological significance. As part of our drug discovery program for antifilarial agents from Indian medicinal plants, the roots of G. glabra were chemically investigated, which resulted in the isolation and characterization of an antifilarial agent, glycyrrhetinic acid (GA, 1a) effective against microfilariae (mf) in vitro (LC100: 12.5 µM; IC50: 1.20 µM), but was inactive against adult worms. Further, GA (1a) was converted into six analogs (2a-7a) and their antifilarial potential was evaluated by studying in vitro motility and MTT reduction assays employing mf and adult worms of Brugia malayi. The results showed that out of six GA analogs, the benzyl amide analog (6a) killed adults and mf at 25 and 50 µM concentration, respectively, and inhibited 49% MTT reduction potential of the adult parasites. The IC50 values were found to be 8.8 and 2.2 µM for adults and mf, respectively. The SI of the compound was >60. On the other hand the octylamide analog (7a) required much higher concentration to adversely affect the parasites. Finally, both active amide analogs (6a and 7a) were in vivo evaluated using B. malayi-jird model, which showed that analog 6a possesses promising macrofilaricidal activity at 100mg/kg, s.c. ×5 days and around 40% of the treated animals showed calcified masses of worm fragments in peritoneal cavity of the animals. To the best of our knowledge this is the first ever report on the antifilarial potential of GA analogs. Further work on optimization of the antifilarial lead is under progress.

Antifilarial diarylheptanoids from Alnus nepalensis leaves growing in high altitude areas of Uttarakhand, India.

Lymphatic filariasis continues to be a major health problem in tropical and subtropical countries. A macrofilaricidal agent capable of eliminating adult filarial parasites is urgently needed. Platyphyllenone (A), alusenone (B), hirustenone (C) and hirsutanonol (D) are important biologically active diarylheptanoids present in Alnus nepalensis. In the present study, we report the antifilarial activity in diarylheptanoids isolated from the leaves of A. nepalensis. Out of four compounds (A-D) tested in vitro one has shown promising anti-filarial activity both in vitro and in vivo studies. This is the first ever report on antifilarial efficacy of a compound of the plant and warrants further studies around this scaffold. In addition, a sensitive, selective and robust densitometric high-performance thin-layer chromatographic method was developed and validated for the above four biomarker compounds. The separation was performed on silica gel 60F(254) high-performance thin layer chromatography plates using chloroform:methanol (9:1, v/v) as mobile phase. The quantitation of marker compounds was carried out using densitometric reflection/absorption mode at 600 nm after post-chromatographic derivatization using vanillin-sulfuric acid reagent. The method was validated for peak purity, precision, robustness, limit of detection (LOD) and quantitation (LOQ) etc., as per the International Conference on Harmonization (ICH) guidelines.

Galactolipids from Bauhinia racemosa as a new class of antifilarial agents against human lymphatic filarial parasite, Brugia malayi.

Bioassay guided fractionation of ethanolic extract of the leaves of Bauhinia racemosa led to the isolation of galactolipid and catechin class of the compounds (1-7) from the most active n-butanol fraction (F4). Among the active galactolipids, 1 emerged as the lead molecule which was active on both forms of lymphatic filarial parasite, Brugia malayi. It was found to be better than the standard drug ivermectin and diethylcarbamazine (DEC) in terms of dose and efficacy.

Gedunin and photogedunin of Xylocarpus granatum possess antifilarial activity against human lymphatic filarial parasite Brugia malayi in experimental rodent host.

The present study is aimed to evaluate antifilarial activity of Xylocarpus granatum (fruit from Andaman) against human lymphatic filarial parasite Brugia malayi in vivo. The in vitro antifilarial activity has already been reported earlier for this mangrove plant which has traditionally been used against several ailments. Aqueous ethanolic crude extract, four fractions (ethyl acetate fraction, n-butanol fraction, water-soluble fraction and water-insoluble fraction) and pure molecule/s of X. granatum (fruit) were tested in vitro on adult worms and microfilariae (mf) of B. malayi and the active samples were further evaluated in vivo in B. malayi (intraperitoneally) i.p. transplanted in the jird model (Meriones unguiculatus) and Mastomys coucha subcutaneously infected with infective larvae (L3). The crude aqueous ethanolic extract was active in vitro (IC50: adult = 15.46 µg/ml; mf = 13.17 µg/ml) and demonstrated 52.8% and 62.7% adulticidal and embryostatic effect on B. malayi, respectively, in Mastomys at a dose of 5 × 50 mg/kg by oral route. The antifilarial activity was primarily localized in the ethyl acetate-soluble fraction which revealed IC50 of 8.5 and 6.9 µg/ml in adult and mf, respectively. This fraction possessed moderate adulticidal and embryostatic action in vivo in Mastomys. Out of eight pure molecules isolated from the active fraction, two compounds gedunin (IC50 = 0.239 µg/ml, CC50 = 212.5 µg/ml, SI = 889.1) and photogedunin (IC50 = 0.213 µg/ml, CC50 = 262.3 µg/ml, SI = 1231.4) at 5 × 100 mg/kg by subcutaneous route revealed excellent adulticidal efficacy resulting in to the death of 80% and 70% transplanted adult B. malayi in the peritoneal cavity of jirds respectively in addition to noticeable microfilaricidalo action on the day of autopsy. The findings reveal that the extract from the fruit X. granatum contains promising in vitro and in vivo antifilarial activity against human lymphatic filarial parasite B. malayi which could be attributed to the presence of two pure compounds gedunin and photogedunin.

Potential antifilarial activity of fruit extracts of Ficus racemosa Linn. against Setaria cervi in vitro.

Effect of alcoholic and aqueous extracts of the fruits of F. racemosa Linn., on the spontaneous movements of both the whole worm and nerve muscle preparation of Setaria cervi and on the survival of microfilariae in vitro was studied. Alcoholic as well as aqueous extracts caused inhibition of spontaneous motility of whole worm and nerve muscle preparation of Setaria cervi characterized by increase in amplitude and tone of contractions. Initial stimulatory effect was not observed with aqueous extract on whole worm preparation, while effect of alcoholic extract on whole worm and nerve muscle preparation was characterized by an increase in amplitude and tone of contractions followed by paralysis. The concentrations required to inhibit the movement of the whole worm and nerve muscle preparation for alcoholic extract of fruits of F. racemosa were 250 and 50 microg/ml, respectively, whereas aqueous extract caused inhibition of the whole worm and nerve muscle preparation at 350 and 150 microg/ml, respectively, suggesting a cuticular barrier. Both alcoholic and aqueous extracts caused death of microfilariae in vitro. LC50 and LC90 were 21 and 35 ng/ml, respectively for alcoholic, which were 27 and 42 ng/ml for aqueous extracts.

Exploration of antifilarial potential and possible mechanism of action of the root extracts of Saxifraga stracheyion on cattle filarial parasite Setaria cervi.

The effect of aqueous and alcohol extracts of the roots of Saxifraga stracheyi Engl. On the spontaneous movements of both the whole worm and the nerve muscle preparation of Setaria cervi and on the survival of microfilariae in vitro was studied. Both extracts caused the inhibition of spontaneous movements of the whole worm and the nerve-muscle preparation of S. cervi, characterized by an increase in the amplitude and a decrease in the rate of contractions, while the tone of the contractions remained visibly unaffected. The concentration required to inhibit the movements of the whole worm preparation was 140 microg/ml for the aqueous and 250 microg/ml for the alcohol extract. The concentration of S. stracheyi extracts required to produce an equivalent effect on the nerve-muscle preparation was 30 microg/ml for aqueous, and 20 microg/ml for the alcohol extract, suggesting a cuticular permeability barrier.

Enhancement of membrane damage by saponins isolated from Acacia auriculiformis.

Acaciaside A and B, two acylated triterpenoid bisglycosides isolated from the funicles of Acacia auriculiformis, are known to have antihelmintic activity. Since the saponins contain a conjugated unsaturated system that is highly susceptible to peroxidation, we investigated the interaction of saponins and membrane using rat liver microsomes as our model. Microsomal membranes were incubated with saponins at 30 degrees C for 2 hr. Following incubation, lipid peroxidation was measured in terms of malondialdehyde and conjugated diene. Our results showed that incubation of microsomal membranes with saponins increased both malondialdehyde and conjugated diene. The results suggest that in our model, saponins enhance the membrane lipid peroxidation.

Antifilarial activity of Mallotus philippensis Lam. on Setaria cervie (Nematoda: Filarioidea) in vitro.

The effect of aqueous and alcoholic extracts of the leaves of Mallotus philippensis (Lam.) Muell. Arg. was studied on the spontaneous movements of the whole worm and nerve-muscle (n.m.) preparation of Setaria cervi and on the survival of microfilariae in vitro. Both the extracts caused inhibition of spontaneous motility of whole worm and the n.m. preparation of S. Cervi characterized by initial stimulation followed by depression in amplitude. The tone and rate of contractions remained visibly unaffected. Aqueous extract at higher concentration showed immediate reduction in tone. The concentration required to inhibit the movements of n.m. preparation was 1/5th for aqueous and 1/11th for alcoholic extract compared to that for the whole worm, suggesting a cuticular permeability barrier. The stimulatory response of acetylcholine was blocked by aqueous extract on whole worm movements. On the microfilariae the LC50 and LC90 were 18 and 20 ng/ml for aqueous and 12 and 15 ng/ml for alcoholic extracts respectively.

Antiparasitic activity of Annona muricata and Annona cherimolia seeds.

Methanolic extracts of Annona muricata and A. cherimolia (Annonaceae) seeds were tested for antiparasitic activity against E. histolytica, N. brasiliensis, M. dessetae and A. salina. The acetogenins isolated from these extracts are found to be responsible for the important activity on infective larvae of Molinema desetae.