Anti-Onchocercal Properties of Extracts of Scoparia dulcis and Cylicodiscus gabunensis

Introduction The elimination of onchocerciasis is hampered by the absence of suitable drugs that are effective against adult filariae. This study is aimed at assessing the anti-onchocercal effects of extracts of Scoparia dulcis and Cylicodiscus gabunensis that could serve as drug leads against onchocerciasis. Methods Different parts of the plants (Scoparia dulcis and Cylicodiscus gabunensis) were extracted with hexane, methylene chloride, and methanol. The extracts were tested in vitro against the bovine model parasite, Onchocerca ochengi. Adult female worm viability was determined biochemically by MTT/formazan colorimetry, while the adult male and microfilariae viability were determined by microscopy based on % inhibition of worm motility score. Cytotoxicity and acute toxicity of active extracts were tested on monkey kidney epithelial cells (LLC-MK2) and Balb/C mice, respectively. Results The hexane extract of Scoparia dulcis recorded the highest activity, with IC50s of 50.78 μg/ml on both adult male and female worms and 3.91 μg/ml on microfilariae. For Cylicodiscus gabunensis extract, the highest activity was seen with the methylene chloride extract, with IC50s of 50.78 μg/ml, 62.50 μg/ml, and 16.28 μg/ml on, respectively, adult male, female, and microfilariae. The 50% cytotoxic concentration on the LLC-MK2 cells was 31.25 μg/ml for the most active extracts. No acute toxicity was recorded for the extracts. Phytochemical analysis of the extracts revealed the presence of alkaloids, flavonoids, sterols, saponins, phenols, and glycosides. Conclusion This study validates the traditional use of these plants in treating onchocerciasis and suggests S. dulcis and C. gabunensis as new potential sources for the isolation of anti-onchocerca lead compounds.


Introduction
Medicinal plants are widely used in traditional cultures all over the world and are becoming increasingly popular in modern society to meet health care needs, particularly in Africa and most developing countries [1]. Natural product molecules represented more than 50% of drugs that had been put into the drug market, with many medicinal plants extracts now used as prescription drugs in many developed countries [2][3][4]. Approximately, a quarter of all Food and Drug Administration and European Medical Agency (EMA) approved drugs are plant-based, with well-known drugs such as quinine, artemisinin, morphine, and the anticancer drugs paclitaxel and vincristine [5][6][7][8]. Te presence of phytoconstituents gives plant species their medicinal potential with numerous functions such as antimicrobial, anticancer, antioxidant, and antiviral [1,9].
Scoparia dulcis is a herb of the family Plantaginaceae that is common in Tropical Africa, Asia, and Central America. It is used traditionally to treat fever, cold, sore throat, and eczema, with other ethnomedical uses like the treatment of gastric problems, reproductive issues, piles, liver, and respiratory diseases [10,11]. Approximately, 160 compounds have been identifed from S. dulcis, among which 155 have been related to the treatment of metabolic syndromes [10]. Previous chemical analysis of the plant has identifed various phytoconstituents such as nitrogen-containing compounds, favonoids, diterpenoids, triterpenoids, steroids, phenolics, and aliphatics, with several pharmacological efects such as been antidiabetics, anticancer, antiarthritic, antihyperlipidaemia, anti-infammatory, and antiurolithiasis [10,12,13].
Cylicodiscus gabunensis (Fabaceae) is an indigenous medicinal plant that is widely distributed in West and Central Africa. Extracts from its stem bark have been used for the treatment of viginitis, jaundice, antimalaria, antibacteria, anti-infammatory, antipyretics, and as soaps and mouthwash by some communities [14][15][16][17]. Previous phytochemical analysis has revealed the presence of triterpenoids, saponins, and phenolics with some defnite pharmacological antimalaria, antifree radical, and antibacteria properties [16,18,19]. Ethnopharmacological information about S. dulcis and C. gabunensis indicated their use by an ethnic group in Cameroon to treat flarial infections like onchocerciasis.
Human onchocerciasis, or river blindness, is a neglected tropical disease with serious debilitating efects, afecting over 37 million people worldwide, mostly in developing countries, including Yemen and many countries found in Africa, Central and South America [20]. Over 200 million people are at risk of onchocerciasis infection, and more than 99% of the disease burden is found in Africa, with an estimated 1.15 million of the infected being blind and an additional 500, 000 living with severe visual impairment [21][22][23]. Te disease is caused by the flarial parasitic nematode Onchocerca volvulus, with the Simulium blackfy being its vector. Despite several eforts to control the disease, limitations are found in new strategies for blocking transmission [24]. Ivermectin remains the only drug currently recommended for the treatment and control of the disease. Te drug mainly targets the microflarial (juvenile) stage of the parasite, leaving the adult worms to continue to reproduce. Tere is also a serious adverse efect in individuals who are treated with ivermectin and are coinfected with Loa loa with high microflaraemia (greater than 30 000 microflariae per ml) [25]. Reports on the resistance of the parasites to some of the flaricidal drugs, coupled with some of their drawbacks and restrictions of use in some coendemic areas, have led to the urgent search for alternative treatments with macroflaricidal properties [7,25]. One strategy employed has been the exploitation of medicinal plants for the identifcation of novel potential drug leads. Tis study investigated the flaricidal properties of Scoparia dulcis (Plantaginaceae) and Cylicodiscus gabunensis (Fabaceae) on cattle derived from Onchocerca ochengi (the closest known relative of Onchocerca volvulus), and their possible use as sources of new drug leads for the treatment of onchocerciasis.

Preparation of Plant Crude Extracts.
Te harvested plants were air-dried and ground to a fne powder. Te ground material was weighed and subsequently immersed and macerated for 72 hours in three diferent solvents, namely: hexane (Hex), methylene chloride (MeC), and methanol (MeOH). For each solvent, the maceration was repeated twice. Te mixture was fltered and the fltrate was concentrated using a rotary evaporator (BUCHI Rotavapor R-200, Switzerland) at appropriate temperatures. Te concentrates were recovered with methylene chloride and allowed to stand at room temperature until the residual solvents had evaporated. Te dried crude extracts were stored at −20°C until needed for assays. Stock solutions of 25 mg/ml of the diferent plant extracts were prepared in >99.8% dimethyl sulfoxide (DMSO) (Sigma, USA) and stored at −20°C until tested in biological assays. Briefy, subcutaneous nodules containing adult O. ochengi worms were identifed on the umbilical skin of infected cows and immediately brought to the laboratory. Under sterile conditions, adult worm masses containing one viable adult female and zero to several adult males were carefully recovered by dissection of the nodule with a sterile razor blade. Te masses were then incubated in 2 ml of complete culture medium (CCM), which comprised RPMI-1640 (Sigma-Aldrich, U.S.A), 10% newborn calf serum, 200 units/ml penicillin, 200 μg/ml streptomycin, and 2.5 μg/ml amphotericin B (Sigma-Aldrich, U.S.A) in standard 12-well culture plates (NUNC, USA) and incubated overnight at 37°C in a 5% CO 2 incubator (Termo Fisher, UK). Te next day, 1000 μg/ml of the diferent plant extracts was prepared in 2 ml of CCM to generate a fnal of 500 μg/ml for the primary screening. Te extracts and controls were tested in triplicate at each concentration, and the experiment was repeated trice on diferent days. Te negative control wells contained only the diluent, DMSO (500 μg/ml). Cultures were terminated on day 7, post addition of plant extracts. Adult male worm viability was visually scored using an inverted microscope (Nikon Eclipse TS200, China) on days 5 and 7, with a percentage reduction of motility ranging from 100% (complete inhibition of motility), 75% (only head or tail of worm motility), 50% (whole body of worm motile but sluggishly), 25% (almost vigorous motility) to 0% (no observable reduction in motility) [26]. Adult female worm viability was assessed on day 7 by the standard MTT/ Formazan assay in which each nodular mass was placed in a well of a 48-well microtitre plate containing 500 μL of 0.5 mg/ml MTT (Sigma-Aldrich, U.S.A) in incomplete culture medium (ICM) (composed of RPMI-1640 (Sigma-Aldrich, U.S.A), 200units/ml penicillin, 200 μg/ml streptomycin, and 2.5 μg/ml amphotericin B, and then incubated in the dark at 37°C for 30 minutes. Adult female worm viability was evaluated visually by the extent to which the female worm mass was stained with MTT. Mean percent inhibition of formazan formation was calculated relative to the negative control worm mass. Adult worm death is positively correlated with the inhibition of formazan formation. Te positive control was auranofn at 30 μM [25]. Secondary screening for the active extracts (100% activity) was done to determine the IC 50 for the extracts. Te active extracts were retested as described under the primary screen at serial dilutions of seven concentrations (from 500 to 7.8125 μg/ml). Assays were done in triplicate and each experiment was repeated for confrmation. Te means of all activities at a concentration were calculated using the statistical analysis Graphpad Prism version 6.0 (Graphpad Software, CA, USA) to generate dose-response curves from which the IC 50 values were obtained.

Screening against O. ochengi Microflariae in Vitro.
Te mammalian kidney cells (LLC-MK2) obtained from the American Type Culture Collection (ATCC, Virginia, USA) were proliferated in CCM at 37°C under an atmosphere of 5% CO 2 in humidifed air. Te cells were seeded in 96-well plates until they became fully confuent and served as feeder layers for the mf assays. Te cells were also used for cytotoxicity assessment of the extract [27]. O. ochengi microflariae were isolated by the method of Cho-Ngwa et al. [28] with slight modifcations. Briefy, umbilical cattle skin pieces containing palpable nodules were obtained from the abattoir, cleaned, carefully shaved, and sterilized with 70% ethanol. Skin slivers were obtained and incubated for 4-6 hours at room temperature in CCM. Te emerged O. ochengi mf was concentrated by centrifugation (400xg, 10 min.). Te highly motile mf was resuspended in CCM and distributed into wells (approximately 15mf/100 μl of CCM/well) of 96well plates containing the LLC-MK2 cell layer, and their viability and sterility were ascertained at the 24th hour prior to the addition of extracts.
Te primary screens for O. ochengi mf were done at 500 μg/ml in duplicates to eliminate inactive extracts. Extracts that showed 100% activity were retested in the secondary screening as described above for the adult worm assay to determine the IC 50 values. Te mf was incubated at 37°C under an atmosphere of 5% CO 2 in humidifed air for 5 days. Te positive control was amocarzine (5 mM), while the negative control contained 2% DMSO. Mf motility was scored microscopically daily. Te percentage motility inhibition scores were assigned as 100% (all mf immotile), 75% (only head or tail of mf shaking occasionally), 50% (whole body of mf motile but sluggishly or with difculty), 25% (almost vigorous motility), and 0% (vigorous motility) [28,29].

Cytotoxicity Studies.
Tis experiment was conducted on the active extracts only. Te fully confuent cells were cultured in the presence of the extracts at varying concentrations and observed daily under an inverted microscope. By day 7, an MTT colorimetric assay was done on the cells [30]. Succinctly, MTT colorimetric assay involved the culturing of LLC-MK2 cells in CCM in a 96-well fat bottom plate, until when the cells attain a density of approximately, 3000 cells/ 100 μl of CCM. Tereafter, the culture media of the fully confuent cells was removed by inverting, ficking, and blotting the plate, followed by the addition of crude extracts in fresh CCM. Colored extracts were washed twice with 100 μL of ICM. A 5 mg/ml MTT stock in 1x phosphate bufer saline was prepared and diluted in ICM to obtain a concentration of 1 mg/ml. To the cells in each well was added 100 μl of the 1 mg/ml solution of MTT. Te plate was incubated for 3 hours at 37°C in a humidifed 5% CO 2 incubator. At the end of incubation, the MTT solution was removed by inverted ficking and blotting of the plate. One hundred microliters (100 μl) of DMSO was added to each well and the plate was shaken at 400 rpm for 5 minutes. Inhibition of formazan formation with MTT directly correlates with cell death, purple formazan formation indicated viable cells, while pale purple correlated with the IC 50 of the cells [30].
Under an inverted microscope (Nikon Eclipse TS200, China), living cells are fattened out and attached to the culture plate, while dead cells are rounded up and detached from the bottom of the plate.
Te selectivity index (SI) values were calculated using the ratio as follows: SI � CC 50 of mammalian cells IC 50 of parasite . (1)

Acute Toxicity Studies.
Te test was conducted in accordance with the OECD guidelines for testing of chemicals [31] and the animal protocol was approved by Te Animal Care and Use Committee, of the Faculty of Science, University of Buea. Te extracts with antiflaricidal activity were tested for acute toxicity in BALB/c mice. A total of 12 animals of approximately 20 g of body weight each were used and divided into 2 groups, 6 for each extract and of equal sex. Each of the 2 treatment groups received one of the extracts at a limited dose of 2000 mg/kg body weight, administered orally in a maximum volume of 250 μl of vegetable oil per animal, while the control group received the diluent only. Te animals were observed daily for 14 days for any changes in physical activity, food intake, and water intake, loss of fur, stool samples, sensitivity to sound, and sensitivity to pain, motility, and mortality.

Statistical
Analysis. Data were analyzed using Graphpad Prism version 6 (Graphpad Software, CA, USA) to determine the IC 50s and mean motility scores.

Results and Discussion
A total of 6 crude extracts were prepared from the two plants using solvents of diferent polarities (hexane, methylene chloride, and methanol). Results of the primary screen showed that the hexane and methylene chloride extracts of the two plants were more efective in killing the adult Onchocerca ochengi male, female, and microflaria with IC 50 values within the range of 62.5-3.91 μg/ml (  (Figures 1(a)-1(c)). Te time-dependent studies of SDHex and SDMeC demonstrated a 100% inhibition of mf motility on the 72 nd hour for both extracts at 7.81 μg/ml, while those of CGHex and CGMeC showed 75% and 100% inhibition, respectively, for mf motility on the 120 th hour at 31.25 μg/ml (Figure 2).

Cytotoxicity and Acute Toxicity of Active Extracts.
Te drug concentration inducing cytotoxicity in 50% of cells (CC 50 ) was 31.25 μg/ml for both extracts of S. dulcis, while the CC 50 for C. gabunensis was 3.0 μg/ml and 6.5 μg/ml for hexane and methylene chloride extracts, respectively. Te Selectivity Index (SI) values of the extracts for adult worms and mf of the parasite ranged from 0.1 to 7.99 (Table 2). In general, the cytotoxicity assay demonstrated that for S. dulcis extracts, the adult worms had SI values below 1, while the mf had SI values above 1. Te SI values for the C. gabunensis extracts were all below 1.
Te active extracts were selected for acute toxicity studies in BALB/C mice at a limited dose of 2000 mg/kg of body weight. Following the administration of the test substance to that of the control mice, no change was observed in the physical activity and behavior of the test and control animals. Te test and control groups were indistinguishable from one another on the basis of their appearance and physical activity at the end of the 14 days study period.

Phytochemical Composition of Active Extracts.
Phytochemical screening revealed the diferent classes of secondary metabolites present in the active hexane and methylene chloride extracts of S. dulcis and C. gabunensis. Te S. dulcis extracts showed positive for the presence of steroids, glycosides, phenolics, and favonoids (Table 3), with alkaloids and saponins present only in the hexane and methylene chloride extracts, respectively. Te C. gabunensis extracts showed positive for the presence of steroids, glycosides, phenolics, and favinoids (Table 3), with alkaloids and saponins present only in the hexane and methylene chloride extracts, respectively.

Discussion
Te aim of this study was to appraise the anti-onchocercal activity of two medicinal plants-Scoparia dulcis and Cylicodiscus gabunensis. From the ethnopharmacological survey, the plants were selected for the study based on their traditional claim to manage flariasis by traditional health practitioners in Okpambe; a village in the Takamanda area in the Manyu Division of the South West Region Cameroon. Tere has not been any scientifc report on the use of these plants and its metabolites for the management of flaria infections. Previous studies of the plants have been reported for their use as antidiabetes, anti-infammatory, antimicrobial, antisickling, antioxidant, antimalarial, and in the treatment of skin diseases, warts, jaundice, dysentery, gonorrhea, snake bites, and gastrointestinal disorders [11,15,33,34]. Onchocerca ochengi was considered the most suitable model for screening anti-onchocerca phytomedicines because it is the closest relative to Onchocerca volvulus in which both parasite shares the same Simulium vector, in addition to the low cost of obtaining O. ochengi and the ease of its availability [25,26].
Te primary screen was done in vitro on all stages of the Onchocerca ochengi parasite for the hexane, methylene chloride, and methanol extracts of S. dulcis and  (Figures 1(a) and 1(b)). Te activity against the adult onchocerca worms is an important fnding because as of date, the lone approved recommended drug for the treatment of the disease does not kill the adult worms (macroflariae), leaving it to continue to reproduce and produces the microflariae (juvenile worms) that generate pathologies [25]. Te hexane and methylene chloride extracts of S. dulcis exhibited the same activity on the microflariae with an IC 50 value of 3.91 μg/ml, while the hexane extract of C. gabunensis recorded an IC 50 value of 22.10 μg/ml (Figure 1(c)). Tese results add information in support of previous fndings that showed nonpolar extracts to be nematocidal more than polar ones [27,35,36]. Te seemingly same IC 50 values of both extracts on the male and female Onchocerca parasites might be due to the fact that both parasites have the same targets for the active plant extracts, while the discrepancy in activity between the adult and juvenile parasites probably indicates a variation in the target of the diferent extracts. Te juvenile form of the parasites was more liable to be killed than the adult worms. Tis might be due to the fact that the juvenile worms are not entangled in nodular masses like adult worms, giving them more exposure to the compounds than adult parasites [29]. In addition, the fact that the extracts could kill the diferent parasitic stages of O. ochengi making these plants a potential source for the isolation of novel compounds with anti-onchocerca activity. Te safety of the plant extracts was done by evaluating cytotoxicity in LLC-MK2 cells and acute toxicity in Balb/c mice. Te selectivity index of S. dulcis extracts was less than 1 for the adult parasites and 7.99 (greater than 1) for the mf, while that of the C. gabunensis extracts was all less than 1. Tese values indicate that the hexane and methylene chloride extracts of S. dulcis and the extracts of C. gabunensis were more toxic to the LLC-MK2 cells with respect to the adult parasites, while the hexane and methylene chloride extracts of S. dulcis were more toxic to the juvenile parasites than the LLC-MK2 cells. Te high toxicity of the extracts on the adult parasites might be due to the complex morphology of the adult parasites relative to the LLC-MK2 cells, where a fairly high concentration of active molecules is required for feasible responses in activity. We observe that none of the laboratory mice died after administration of the active extracts at a limited dose during acute toxicity studies. Tis fnding corroborates previous toxicology studies of these plant extracts, which demonstrated no toxicity to laboratory rats [11]. Te use of these plants in the treatment of flarial and other infections by the indigenes of Okpambe and other indigenous populations over centuries without serious side efects is proof of their safety. Te absence of adverse efects in patients who take the plant concoctions for therapy might be due to in vivo detoxifcation mechanisms, which are absent in in vitro studies.
Phytochemical analysis of S. dulcis and C. gabunensis revealed the presence of secondary metabolites; favonoids, sterols, phenols, and glycosides in both extracts of the plants,   and alkaloids in the hexane extracts and saponins in the methylene chloride extracts. Tis suggests that the active principles in the extracts may be from the groups of compounds mentioned above. Tis is in conformity with previous phytochemical studies, which revealed the presence of these secondary metabolites to be responsible for the medicinal properties of these plants [16,33]. Te isolation of pure compounds from these plant extracts is recommended to determine their full active potential and ascertain the actual phytochemicals responsible for the anti-onchocerca activity of the plant extracts. Such refnement might enhance the activity of the lead compounds against the parasite, and possible reaction mechanisms could be elucidated from any pure lead compound isolated with an attribute of anti-onchocerca activities.
Remarkably, none of the S. dulcis and C. gabunensis extracts have been previously tested against Onchocerca and other flaria species. Our data reinforce the existing knowledge and regular use of these plants by traditional health practitioners for the treatment of infectious diseases.

Conclusions
Tis study has revealed for the frst time the anti-onchocercal activity of extracts of S. dulcis and C. gabunensis, which could serve as potential novel sources of new drugs against onchocerciasis. It also validates the use of these plants by traditional health practitioners in the local management of flariasis.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te author declares they have no conficts of interest.

Authors' Contributions
Fidelis Cho-Ngwa conceptualized the study. Tiku Edward Tiku and Moses Samje curated the data. Napoleon Mfonku investigated the study. Fidelis Cho-Ngwa and Samje Moses collected the resources. Fidelis Cho-Ngwa supervised the study. Tiku Edward Tiku visualized the study. Tiku Edward Tiku and Samje Moses written the original draft. Tiku Edward Tiku, Samje Moses, and Fidelis Cho-Ngwa edited and reviewed the manuscript.