Antihelminthic Activity of Lophira Lanceolata on Heligmosomoides polygyrus Using an Automated High-Throughput Method

Background There are about 13 parasitic infections that are responsible for significant morbidity and mortality but have not received the attention they deserve; thus, they are now known as “neglected tropical diseases” (NTDs). This study was aimed at evaluating the antihelminthic activities of Lophira lanceolata using an automated high-throughput method. Methods The antihelminthic activity effect of the extracts against H. polygyrus was determined using an automated high-throughput method. For the egg-hatching test, 100 μL of embryonated egg suspension (60 eggs) was added to 100 μL of various concentrations of extracts, levamisole, and 1.5% DMSO in a 96-well round-bottom microtitre plate. After mixing, the 96-well microplate was placed in WMicroTracker and incubated for 24 h at 25°C; the movements were recorded every 30 minutes. The same procedure was used for the larval motility assays, where 100 μL of L1 or L2 larvae (50 larvae) were put in contact with 100 μL of various concentrations of extracts. Results The ovicidal activity (hatching) had an IC50 of 1.4 mg/mL for the ethanol extract. The aqueous and ethanol extracts of L. lanceolata showed larvicidal activity on the L1 larvae with IC50 of 1.85 mg/mL and 2.4 mg/mL, respectively, as well as on the L2 larvae with IC50 values of 1.08 mg/mL and 1.02 mg/mL for the aqueous and ethanol extracts, respectively. These results showed that the aqueous extract exhibited a stronger inhibitory power on the hatching rate of parasites than ethanol extracts, while the contrary effect was observed for the larval motility assays. Conclusion This study provides scientific data on the use of L. lanceolata by the local population for the treatment of helminthiases. However, in vivo and toxicity tests are necessary to assess its activity and safety.


Introduction
Helminthiasis is one of the most devastating diseases in most developing countries in Africa. Most of the research funding for the control of diseases is directed toward HIV and tuberculosis [1]. Little or no attention is paid to helminth infections; hence, they are called neglected tropical diseases (NTDs). According to a recent study conducted by Cedric et al. [2], a prevalence of 23.5% was observed amongst children in Bambili, Northwest Region of Cameroon. Similar observations were performed by Igore et al. [3], where they had observed a prevalence of 26.4% in Tonga subdivision, West Region of Cameroon. All these high prevalence rates indicate that Cameroon has a serious problem of helminthiases.
Most of the helminth parasites are showing resistance to available synthetic drugs. Furthermore, these drugs have side efects and are not easily available to the local population [4]. In Cameroon, the use of herbal medicines for the treatment of several ailments including helminthiases is very common [5]. Te local population of the west region of Cameroon usually relies on medicinal plants to overcome this problem of resistance, availability, and high cost of these synthetic drugs. Resistance to antihelminthic drugs is on the rise, in part due to their injudicious use [6]. It is therefore of paramount importance to rapidly screen medicinal plants used by the local population to treat helminthiases in order to justify their usage by the local population. Furthermore, little is known about the optimum dose and treatment frequency of such plants, as well as potential toxicities. Terefore, alternative control strategies are urgently needed to validate these medicinal plants.
Lophira lanceolata is a medicinal plant used in traditional medicine to treat many diseases. Te roots are used to treat wounds, amenorrhoea, sterility, constipation, diarrhoea, jaundice, and vomiting [7]. Te bark is used to treat kwashiorkor, jaundice, acute hepatitis, enteritis, coughs, and bronchitis [8]. Te twigs are used to treat gingivitis, hypertension, dysentery, and syphilis. In the dry regions of West Africa, the oil extracted from the seeds is used as a body balm against lice and leprosy. Te oil is used for many medico-religious purposes [9]. Lophira lanceolata is used by the local population of the western region of Cameroon for the treatment of helminthiasis.
Most conventional in vitro screening methods for parasitic worms have had limitations in that most of them are time-consuming to perform and/or lack repeatability/ reproducibility [10].
Te WMicroTracker (Phylumtech, Argentina), is a rapid, automated high-throughput equipment used for the evaluation of the antihelminthic activity of drugs by sending infrared beams to worms and recording the movement of worms [11]. Heligmosomoides polygyrus is a parasitic nematode (as opposed to nonparasitic nematodes such as Caenorhabditis elegans) used as a model for the evaluation of the antihelminthic activity of drugs. Studies carried out using H. polygyrus in this domain include those of Teufack et al. [12], Poné et al. [13], and Komtangi et al. [14]. Tis paper shows for the frst time that H. polygyrus is used with the WMicroTracker system for the evaluation of the antihelminthic activity of a medicinal plant. Te aim of this study was to evaluate the antihelminthic activity of Lophira lanceolata, a medicinal plant used for the treatment of helminthiasis in Foumban, West Region of Cameroon, and provide scientifc evidence on its use as an antihelminthic agent.

Helminth Parasite.
Te infective third-stage larvae (L3) of H. polygyrus were generously provided by Pr. Rick Maizels, University of Edinburgh, UK. Te parasite was cultured from the egg to the L2 stage in Petri dishes containing wet flter paper. It is a strongylid nematode related to human hookworm species. Heligmosomoides polygyrus is a standard experimental model used for routine screening of potential drug candidates [15].

Collection and Identifcation of Plant Materials.
Te leaves of Lophira lanceolata were harvested from the peripheral Savannas of Foumban in the Noun Division of the West Region of Cameroon. Te plant leaves, fowers, and fruits were sent to the National Herbarium of Cameroon (NHC) for authentifcation, and it was registered and given the registration number 3512/SRFK-CAM.

Preparation of Extracts.
Te aqueous extract was prepared by infusion, while the ethanol extract was obtained using the procedure described by Poné et al. [13]. Briefy, 100 g of powder was weighed using an electronic balance (SF-400) and introduced into a 5 L container. One liter (1 L) of 95% ethanol was added, and the mixture was stirred for 5 minutes. Te mixture was macerated for 72 hours at room temperature, and every 24 hours, this mixture was stirred. Te homogenate was frst fltered using a 150 μm sieve and then with Whatman paper N°1. Te fltrate obtained was introduced into an oven at 40°C for evaporation until the dry extract was obtained. Te same procedure was performed for infusion except that water was heated at 100°C and that the powder was added.

Collection and Concentration of H. polygyrus Eggs.
Freshly passed out faeces of monoinfected mice (Mus musculus) with a resistant helminth (H. polygyrus) was collected using teaspoon. 5 g of faeces was homogenized in a mortar using a pestle with 60 mL of saturated NaCl solution. Tis solution was successively fltered using a tea sieve and then a 150 μm sieve. Te fltrate was used to obtain H. polygyrus eggs through the fotation technique. Te eggs were centrifuged three times for 10 minutes at 1500 rpm, which enabled us to wash all salt from the eggs. After centrifuging for 10 minutes, the solution was then siphoned using a pipette, distilled water was added, and the solution was centrifuged again.

Coproculture and Collection of H. polygyrus Larvae.
Te eggs obtained above were cultured by incubating them in a Petri dish. Te Petri dish was incubated at 25°C at a relative humidity (RH) of 78% for 2 and 4 days, respectively, with the aim of obtaining L1 and L2 larvae.

Egg-Hatching Test.
Te antihelminthic activity of the extract was determined using the method described by Poné et al. [13] with slight modifcations. Briefy, 100 μL of the embryonated egg suspension containing 60 eggs was incubated with 100 μL of various concentrations of extracts, levamisole, and 3% DMSO in 96-well cell culture roundbottom microtitre plates. Te round-bottom microtitre plates were used so that WMicroTracker could directly send laser beams to worms. Te fnal concentrations in the test plates varied from 5 to 0.3125 g/mL (1.5% DMSO) for the extracts and 5 μg/mL for levamisole in a fnal volume of 200 μL. Te 96-well microplates were incubated for 24 h at 25°C at a relative humidity (RH) of 78% in WMicroTracker, where the worm movements were measured by using WMicroTracker every 30 minutes. Tree independent experiments for the ethanol and aqueous extracts were carried out, and the average results were recorded. Te inhibition percentages of hatching of each sample were determined at the end of incubation, using the average movement over 24 h in the presence of test samples with the extract, compared with the controls, DMSO 1.5% and levamisole [16]:

Larval Motility Assays.
For the efects of the extracts on L1 and L2 larvae, the same procedure described above was followed. Briefy, 100 μL of the L1 larval stage suspension containing 50 L1 larvae was incubated with 100 μL of various concentrations of extracts, levamisole, and 1.5% DMSO in 96-well cell culture round-bottom microtitre plates. Te 96well microtitre plates were incubated in WMicroTracker at 25°C for 24 h; the worm movements were recorded every 30 min. Te larval motility activity was determined by calculating the inhibition percentages [6]. Te same method was used to test the activity of active compounds on the L2 larvae.

Phytochemical
Screening. Te extracts were tested for the presence of sterols, alkaloids, triterpenoids, saponins, anthocyanins, and anthraquinones using standard procedures described by Harbone [17].   Figure 1 shows the percentage of hatching of the aqueous and ethanol extracts. It follows from the analysis of the fgure that hatching was signifcantly inhibited at 5 mg/mL where we observed 5.0 ± 5.0% and 11.66 ± 2.8% for the aqueous and ethanol extracts, respectively. Distilled water and 1.5% DMSO had 100% hatching, indicating that the plant extract had an efect. Te highest percentage of hatching for the extracts was observed with the ethanol extract at concentrations of 0.3125 mg/mL and 0.625 mg/mL with hatching percentages of 65 ± 8.6% and 76.66 ± 15.2%, respectively. Figure 2 shows the IC 50 of the diferent extracts on the hatching rates of H. polygyrus. It follows from the analysis of the fgure that the IC 50 for the ethanol extract was 1.4 mg/mL and that for aqueous was undetermined because it was very wide, showing that the ethanol extract exhibited a stronger inhibitory power on the hatching rate of parasites. Figure 3 shows the inhibition percentage of the aqueous and ethanol extracts on L1 larvae of H. polygyrus. It appears from the fgure that the mean inhibition rate at 5 mg/mL completely inhibited (100%) the mobility of the L1 larvae as compared to the positive control (levamisole) that had a mean inhibition rate of 96.67 ± 2.88. Te ethanol and aqueous extracts had a mean inhibition rate of 96.66 ± 6.44 and 86.66 ± 5.77 at 2.5 mg/mL, respectively. Both distilled water and 1.5% DMSO (negative control) had the mean inhibition percentages of 0%, indicating that the negative controls had no inhibitory efect on the larvae. Te ethanol extract showed a higher inhibitory efect than aqueous extracts. Both the extracts showed a signifcant concentration-dependent efect. Teir efects decreased with a decrease in concentration from 5 mg/mL to 0.3125 mg/mL. Figure 4 shows the IC 50 of the diferent extracts on the frst larval stage of H. polygyrus. From the analysis of the fgure, the IC 50 for both the extracts was 1.85 mg/mL and 2.4 mg/mL, respectively. It follows from the analysis of the IC 50 that the ethanol extract was more active than the aqueous extract. Figure 5 shows the inhibition percentage of the aqueous and ethanol extracts on L2 larvae of H. polygyrus. It follows from the analysis of the fgure that concentration 5 mg/mL had the highest mean inhibition rate of 93.33 ± 5.77 and 100 ± 0.0, respectively, for the aqueous and ethanol extracts, with a signifcant diference. Te aqueous and ethanol extracts at the same concentration inhibited the L2 larvae with a signifcant diference. Distilled water and 1.5% DMSO (negative control) had no efects, while levamisole had 100% inhibition. Te lowest inhibitory activity was observed at 0.3125 mg/mL with inhibitory percentages of 33.33 ± 3.21% and 90 ± 10% for the aqueous and ethanol extracts, respectively. Figure 6 shows the IC 50 of the diferent extracts on the second larval stage of H.polygyrus. Te analysis indicated that the IC 50 for the aqueous extract was 1.08 mg/mL, while that for the ethanol extract was 1.02 mg/mL, indicating that the ethanol extract was more active than the aqueous extract. Table 1 shows the phytochemical screening of Lophira lanceolata extracts. It follows from the analysis of the table that the aqueous extract contains more classes of chemical constituents.

Discussion
Tis study aims to evaluate the antihelminthic activity of L. lanceolata, a medicinal plant used for the treatment of helminthiasis in Foumban, West Region of Cameroon, and provide scientifc evidence on its use as an antihelminthic agent.
Te ovicidal activity (hatching) had an IC 50 of 1.4 mg/ mL for the ethanol extract. Tese results show that the ethanol extract was more active than the aqueous extract. Similar fnding was obtained by Poné [13] with extracts of Ageratum conyzoides on embryonated eggs of H. bakeri. Egg hatching could have been inhibited by saponins in the extracts, as these molecules are known to stop nematodes from egg hatching [18]. Te plant extracts inhibit the development of structures required for hatching in H. Bakeri such as     a protractible stylet mouthpart that is used for both infection and opening of the eggshell during hatching [19]. Moreover, the plant extract may have stopped the exchange of water and sugars across the eggshell, altering the osmotic pressure and the size of the egg, thereby inhibiting the hatching process [20]. Te aqueous and ethanol extracts of L. lanceolata showed larvicidal activity on the L1 larvae with an IC 50 of 0.209 ± 0.05 mg/mL and 0.0033 ± 0.0060 mg/mL, respectively, as well as on the L2 larvae with an IC 50 of 1.85 mg/ mL and 2.4 mg/mL, respectively, as well as on the L2 larvae with IC 50 values of 1.08 mg/mL and 1.02 mg/mL, respectively. Te controls (distilled water and 1.5% DMSO) did not kill the larvae. Te extracts of L. lanceolata possessed larvicidal properties, with the ethanol extract being the most efective one. Similar results were obtained by Kalmobé et al. [21], where the ethanol and the MeOH-CH 2 Cl 2 extracts of the leaves of L. lanceolata showed activity with higher IC 50 values on C. elegans wild-type strains compared to ivermectin, levamisole, and albendazole after 72 h of incubation.
Tis confrms the fact that the dead larvae found inside the treated 96-well microplates were due to the active ingredients of the plant extracts, thus indicating their larvicidal properties. Tis observation was also reported by Gertrude et al. [22] when evaluating in vitro ovicidal and larvicidal activities of the aqueous and ethanol extracts of the leaves of Bidens pilosa (Asteraceae) on Heligmosomoides bakeri (nematoda: Heligmosomatidae).
Troughout this study, we observed that L1 larvae were more vulnerable to diferent plant extracts than L2 larvae. Te larvicidal properties of these extracts may be due to the penetration of active compounds across the cuticle of the larvae on one hand or the absorption of the substance by the larvae. Tis fnding is similar to that obtained by Payne [23]. Payne [23] mentioned that active compounds penetrate the cuticle of nematodes and prevent the absorption of glucose or block postsynaptic receptors, thus paralyzing the larvae. Te active compound may inhibit the transmission of nervous impulses by gamma-aminobutyric acid (GABA) whose secretion is stimulated by the active compound [23]. Lem et al. [24] mentioned that active compounds found in food can cross the intestinal lining of larvae and gain access to the circulatory system of the organism. Moreover, active compounds like tannin may bind to the cuticle of the nematode, destabilize the membrane, and increase cell permeability by combining with membrane-associated sterols which lead to death [25].

Conclusion
Te results obtained in our study scientifcally validate the use of L. lanceolata in fghting against helminthes in Cameroonian folk medicine. However, further studies to determine its safety in order to assess its toxicity are needed to establish it as an antiparasitic agent.

Data Availability
All the data generated and analysed are included in this research article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.