Anthelmintic Activity and Cytotoxic Effects of Compounds Isolated from the Fruits of Ozoroa insignis Del. (Anacardiaceae).

Ozoroa insignis Del. is an ethnobotanical plant widely used in traditional medicine for various ailments, including schistosomiasis, tapeworm, and hookworm infections. From the so far not investigated fruits of Ozoroa insignis, the anthelmintic principles could be isolated through bioassay-guided isolation using Caenorhabditis elegans and identified by NMR spectroscopic analysis and mass spectrometric studies. Isolated 6-[8(Z)-pentadecenyl] anacardic (1), 6-[10(Z)-heptadecenyl] anacardic acid (2), and 3-[7(Z)-pentadecenyl] phenol (3) were evaluated against the 5 parasitic organisms Schistosoma mansoni (adult and newly transformed schistosomula), Strongyloides ratti, Heligmosomoides polygyrus, Necator americanus, and Ancylostoma ceylanicum, which mainly infect humans and other mammals. Compounds 1-3 showed good activity against Schistosoma mansoni, with compound 1 showing the best activity against newly transformed schistosomula with 50% activity at 1µM. The isolated compounds were also evaluated for their cytotoxic properties against PC-3 (human prostate adenocarcinoma) and HT-29 (human colorectal adenocarcinoma) cell lines, whereby compounds 2 and 3 showed antiproliferative activity in both cancer cell lines, while compound 1 exhibited antiproliferative activity only on PC-3 cells. With an IC50 value of 43.2 µM, compound 3 was found to be the most active of the 3 investigated compounds.

Hydro-Ethanolic Extract of Mentha pulegium Exhibit Anthelmintic and Antioxidant Proprieties In Vitro and In Vivo.

INTRODUCTION: During recent decades, the emergence of chemoresistance among synthetic anthelmintic drugs has increased the interest in screening novel natural anthelmintic compounds derived from plants. The current study is aimed to determine the chemical profile, anthelmintic and antioxidant properties of Mentha pulegium hydro-ethanolic extract. MATERIALS AND METHODS: Two tests were used to assess the in vitro anthelmintic activity of the hydro-ethanolic extract of M. pulegium against Haemonchus contortus; egg hatch assay (EHA) and adult worm motility (AWM) assay. M. pulegium extracts at the doses of 500, 1000, 2000 and 4000 mg/kg were evaluated in vivo in mice infected with Heligmosomoides polygyrus. The anthelmintic efficacy was monitored using faecal egg count reduction (FECR) and total worm count reduction (TWCR). The antioxidant activity of M. pulegium extract was evaluated by testing the total antioxidant capacity and the DPPH free radical-scavenging ability. RESULTS: Chromatographic characterization of M. pulegium composition using RP-HPLC revealed the presence of phenolic acids such as syringic acid, ferulic acid and the presence of flavonoid compounds, such as isorhamnetin-3-O-glucoside and kaempferol-3-O-rutinoside. We observed 91.58% inhibition in the EHA at 8 mg/mL after 48 h of incubation (IC50=1.82 mg/mL). In the AWM assay, M. pulegium extract achieved 65.2% inhibition at 8 mg/mL after 8 h. The highest dose (4000 mg/kg) showed a significant nematicidal effect 7 days post-treatment by inducing 60.39% FECR and 71.6% TWCR. We also report strong in vivo antioxidant capacity of the extract, as revealed by a significant increase of the enzymatic activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzymes in mice infected with H. polygyrus. CONCLUSION: Together, the results in this paper suggest that M. pulegium possesses anthelmintic properties and could be a potential source of novel compounds for the control of helminth parasites as well as its associated oxidative damage.

Chamomile Methanolic Extract Mitigates Small Bowel Inflammation and ROS Overload Related to the Intestinal Nematodes Infection in Mice.

BACKGROUND: Chamomile (Matricaria recutita L.) is a plant which has been reported to be effective in treating several parasitic and digestive diseases. The present study was conducted to evaluate the anthelmintic activity of chamomile methanolic extract (CME). METHODS: In vitro, the anthelmintic activities of CME were investigated on the L3 larvae of Heligmosomoides polygyrus in comparison to albendazole. In vivo, Swiss albino mice were infected with infective third (L3) larval stage of H. polygyrus by intragastric administration. Moreover, the effect of CME and albendazole on worm eggs, adult worms, serum cytokine production, and oxidative stress was studied. RESULTS: All used doses of CME showed a potent anthelmintic activity both in vitro and in vivo and the effect being similar to treatment with albendazole. Moreover, H. polygyrus infestation was accompanied by an intestinal oxidative stress status characterized by an increased lipoperoxidation, a depletion of antioxidant enzyme activity, as well as an overload of hydrogen peroxide. We have also recorded an increase of pro-inflammatory mediator (TNF-α, IL-6, and IL-1ß) levels after treatment with CME (14 ± 0.8; 41 ± 2; 58 ± 4 pg/mg protein, respectively, with the concentration 800 mg/kg, body weight) when compared with infected control mice (20 ± 1; 59 ± 2, and 83 ± 4 pg/mg protein, respectively). However, extract treatment alleviated all the deleterious effects associated with H. polygyrus infection. CONCLUSION: These findings suggest that CME can be used in the control of gastrointestinal helminthiasis and associated oxidative stress.

Anthelmintic activity of Securidaca longepedunculata (Family: Polygalaceae) root extract in mice, in vitro and in vivo.

OBJECTIVE: To elucidate the pharmacological bases of oral administration of Securidaca longepedunculata (S. longepedunculata) root extract as an anthelmintic in folkloric medicine. METHODS: Albino mice were infected with infective third (L3) larval stage of Heligmosomoides polygyrus (H. polygyrus) by esophageal intubation. Following establishment of the adult worms in the intestine, the mice were treated with 0-2 000 mg/kg body weight (bw) of methanolic root extract of S. longepedunculata and 100 mg/kg bw of pyrantel embonate, the reference drug in vivo. Bioactivity and larvicidal effects of the extract were tested by exposing brine shrimps (Artemia salina) to 0.00-1.00 mg/mL and the L3 stage of Heligmosomoidescontortus (H. contortus) and H. polygyrus to 0.00-2.50 mg/mL of the extract in vitro. RESULTS: The percentage yield of the extract was 7.13% w/w dry matter. The brine shrimps toxicity bioassay resulted in an LC50 of 74.18 µg/mL. The extract had a significant, dose-dependent larvicidal effect on the L3 stage of H. contortus and H. polygyrus with the terminal effect of 75% and 70% at the highest exposure concentrations, respectively. The extract however, did not affect the number of worm eggs per gram (epg) of fecal materials (P<0.05) and total worm burden (twb) of adult H. polygyrus in infected mice. Treatment with pyrantel embonate significant reduced both the fecal egg count and twb to 0 compared to the untreated control (P<0.05). CONCLUSIONS: These results indicate that S. longepedunculata root extract contains potent bioactive compounds and has larvicidal effect on L3 stage of H. contortus and H. polygyrus, substantiating its use as anthelmintic in alternative medicine.

Changes in Heligmosomoides polygyrus glycoprotein pattern by saponins impact the BALB/c mice immune response.

Saponins of marigold (Calendula officinalis), in particular derivatives of 3-O-monoglucuronide of oleanolic acid, are able to reduce infectivity of Heligmosomoides polygyrus in mice. The purpose of this study was to understand the immune activation provoked by third-stage larvae exposed to marigold glucuronides. We also examined the pattern of glycosylation of larval antigens which appeared to be crucial for induction of cytokine production in BALB/c mice; higher concentrations of IL-6, IFN-γ, IL-10 and TNF-α were observed in serum or intestine one week post infection. Three weeks later, in the chronic phase of infection, cells in culture were able to produce IL-6, IFN-γ, TNF-α and IL-17. Restimulation of cells with H. polygyrus antigen resulted in reduced production of IL-6, and TNF-α. The pattern of cytokine production co-existed with reduced expression of terminal glucose, α-linked mannose, N-acetyl-galactosamine, ß-galactose, N-acetyl-glucosamine and α-fucose in several protein bands. Galactose, as a new terminal carbohydrate residue appeared in 20-24kDa protein bands. The number of immunogenic epitopes in parasitic antigens was reduced; only three protein bands of 56, 26 and 12kDa were recognized by IgG1. These studies provide a model system to find the glycosylated molecules expressed on nematodes that improve establishment and survival and characterize cytokine production in mice infected with larvae exposed to saponin. Identification of these molecules is the first step in the recognition of key antigenic epitopes able to induce protective or tolerogenic immune responses.

In vitro nematicidal activity of extracts of Canthium mannii (Rubiaceae), on different life-cycle stages of Heligmosomoides polygyrus (Nematoda, Heligmosomatidae).

The increasing prevalence of anthelmintic-resistant strains of helminths, drug residues in animal products and high cost of conventional anthelmintics has created an interest in studying medicinal plants as an alternative source of anthelmintic. The potential nematicidal activities of four extracts from the bark of Canthium mannii (Rubiaceae) stem were investigated in vitro. Extracts were diluted in distilled water (DW) to obtain five different concentrations (1.5, 2.0, 2.5, 3.0 and 3.5 mg/ml) and put in contact with eggs and larvae of Heligmosomoides polygyrus. The different stages of the life cycle were also put in contact with the same concentration of mebendazole (MBZ, positive control). One millilitre of each extract at different concentrations and control were added to 1 ml solution containing 30-40 eggs or 10-15 larvae (L1, L2 and L3) and distributed in different Petri dishes. The eggs and larvae were incubated at 24 degrees C and exposure times were: 48 h for un-embryonated eggs, 6 h for embryonated eggs; 2, 4, 6 and 24 h for L1 and L2 larvae, 24-48 h for infective larvae (L3), and 5 days for the larval development test (from L1 to L3). DW and 1% dimethyl sulphoxide (DMSO) were used as placebo and DMSO control, respectively. Significant effects were obtained with three of the four extracts, and differences were observed depending on the parasite stage. Cold water extract (CWE), hot water extract (HWE) and ethanol extract (ETE) inhibited embryonic development (40, 45 and 10%) and hatching of embryonated eggs (40, 85 and 80%), respectively, at 3.5 mg/ml. Only ETE killed L1 (97.18%) and L2 (92.68%) larvae of H. polygyrus after 24 h at 3.5 mg/ml and drastically reduced the production rate (6% at 3.0 and 3.5 mg/ml) of infective larvae (L3) after 5 days of incubation compared to other extracts (P < 0.05). However, the infective larvae of H. polygyrus were resistant to the effect of each of the tested products (extracts and mebendazole). These in vitro results suggested that extracts of C. mannii, used by traditional healers in Dschang, Western Region of Cameroon (Central Africa) to cure intestinal helminthiasis and abdominal pains of their patients, possess nematicidal properties. The active principles responsible for the activity could be secondary metabolites such as alkaloids and saponins present in the extracts. It is suggested that further experiments incorporating in vivo purification of extracts and toxicological investigations should be carried out.

The anthelmintic efficacy of plant-derived cysteine proteinases against the rodent gastrointestinal nematode, Heligmosomoides polygyrus, in vivo.

Gastrointestinal (GI) nematodes are important disease-causing organisms, controlled primarily through treatment with synthetic drugs, but the efficacy of these drugs has declined due to widespread resistance, and hence new drugs, with different modes of action, are required. Some medicinal plants, used traditionally for the treatment of worm infections, contain cysteine proteinases known to damage worms irreversibly in vitro. Here we (i) confirm that papaya latex has marked efficacy in vivo against the rodent gastrointestinal nematode, Heligmosomoides polygyrus, (ii) demonstrate the dose-dependent nature of the activity (>90% reduction in egg output and 80% reduction in worm burden at the highest active enzyme concentration of 133 nmol), (iii) establish unequivocally that it is the cysteine proteinases that are the active principles in vivo (complete inhibition of enzyme activity when pre-incubated with the cysteine proteinase-specific inhibitor, E-64) and (iv) show that activity is confined to worms that are in the intestinal lumen. The mechanism of action was distinct from all current synthetic anthelmintics, and was the same as that in vitro, with the enzymes attacking and digesting the protective cuticle. Treatment had no detectable side-effects on immune cell numbers in the mucosa (there was no difference in the numbers of mast cells and goblet cells between the treated groups) and mucosal architecture (length of intestinal villi). Only the infected and untreated mice had much shorter villi than the other 3 groups, which was a consequence of infection and not treatment. Plant-derived cysteine proteinases are therefore prime candidates for development as novel drugs for the treatment of GI nematode infections.

Activity of the cyclic depsipeptide emodepside (BAY 44-4400) against larval and adult stages of nematodes in rodents and the influence on worm survival.

The present investigations deal with the activity of the cyclic depsipeptide emodepside (BAY 44-4400) against larval and adult stages of three rodent nematodes. While emodepside acts strongly against the adult stages of the rat nematodes Nippostrongylus brasiliensis and Strongyloides ratti, as well as against the mouse nematode Heligmosomoides polygyrus, its actions against the larval stages of these nematodes vary according to the species. Thus, emodepside is highly effective against the lung and intestine larval stages of N. brasiliensis and S. ratti. By contrast. the larval stages of H. polygyrus in the intestine are only partly affected by higher emodepside dosages.

The in vitro secretion of acetylcholinesterase by adult stages of Heligmosomoides polygyrus: the effects of broad-spectrum anthelmintics.

The secretion of acetylcholinesterase (AChE) by female and male Heligmosomoides polygyrus was studied in different in vitro culture media. AChE secretion was increased in the presence of fetal calf serum or bovine serum albumin (BSA). In the absence of crowding effects, specific AChE activity in excretion/secretion products was higher for male (2.41 +/- 0.07 mumol min-1 l-1 mg-1) than for female (0.56 +/- 0.04 mumol min-1 mg-1) worms but on a per nematode basis both sexes showed comparable rates of secretion. Acetylthiocholine iodide was the favoured substrate of the enzyme. When the nematodes were incubated in vitro with albendazole (ABZ), ricobendazole (RBZ), mebendazole (MBZ), levamisole (LVM), morantel (MRT) or ivermectin (IVM), at concentrations from 1 mM to 10 nM, in RPMI medium for 2 or 6 h and then transferred to a drug-free medium (RPMI medium supplemented with 0.5% BSA) for 24 h or continuously exposed to the drugs in supplement-free medium (24 h), the concentration- and time-dependent inhibitory effects on AChE secretion were observed. The continued exposure to the drugs for all incubation periods (with a single exception for LVM 1 mM) produced the highest levels of inhibition. Under these conditions, the concentrations inhibiting the secretion of AChE by 50% (IC50) relative to drug-free controls were estimated. The IC50 values ranged from 0.012 microM (IVM) to 2.96 microM (MRT). The potential of this bioassay for the selective primary evaluation of new compounds with broad-spectrum anti-nematodal activity is discussed.

Simple micromotility recorder for rapid screening of potentially anthelmintic compounds.

A simple micromotility recorder for monitoring the motility of small nematodes (adult) is described. Normal motility of Ancylostoma ceylanicum and Nematospiroides dubius was recorded. The time course to cause paralysis (paralysis time) was also observed in the presence of various anthelmintics, e.g., Levamisole, Pyrantel; and Ivermectin at graded concentrations. This is a simple in vitro model for screening of potential anthelmintic compounds.