Comparative Evaluation of the In Vitro Anthelminthic Effects of the Leaves, Stem, and Seeds of Carica papaya (Linn) Using the Pheretima posthuma Model

This study was conducted to comparatively assess the anthelminthic activity of leaves, stem bark, and seeds of Carica papaya, in order to identify which of the plant parts possess the highest anthelminthic activity. Three concentrations of ethanolic and hydroethanolic extracts of the plant parts (1 mg/ml, 2.5 mg/ml, and 5 mg/ml) were prepared and tested against Pheretima posthuma using albendazole as the positive control and 0.9% normal saline solution as the negative control. Preliminary phytochemical investigation showed the presence of alkaloids, saponins, and reducing sugars of glycosides present in all the crude extracts of Carica papaya. Tannins were observed only in extracts of the leaves, while fixed oils were only present in the extracts of the seeds. The results of the anthelminthic activity testing indicated that all crude extracts prepared were more effective than albendazole in reducing paralysis time (p < 0.0001) and death time (p < 0.0001). It was further shown that the extracts from the seeds (SE and SHE) were more effective than the extracts from the stem bark and leaves both in reducing paralysis and death times. Fractionation of SE provided a fraction, SEB, which was more effective than SE both in reducing paralysis and death times (p < 0.0001) and was established to contain fixed oils. The outcome of the current study has provided a scientific justification for the preference of the seeds of Carica papaya for the treatment of helminth infections and has shown that the fixed oils present in the seeds could be responsible for such activity.


Introduction
It is generally held that helminth infections are the most commonly encountered infections in man [1], with an estimation of approximately three million people infected globally [2]. When an individual is infected, the person excretes faeces infected with helminth eggs, thereby contaminating the soil [1]. Helminth infections usually result in cases of anaemia, pneumonia, eosinophilia, and malnutrition [1]. Other reported symptoms include abdominal pains, nausea, and diarrhoea [3]. Although the infections are not known to be lethal as compared to other infections, they are thought to be recurrent among poor people living in poor sanitary conditions where due to financial constraints are unable to access effective conventional medications [4], and for that reason pose an enormous impact economically on human livelihood [5].
Over the years, conventional anthelminthic drugs, such as, albendazole, mebendazole, ivermectin, piperazine, and niclosamide, have been widely patronized to treat helminth infections, in both livestock and humans. However, due to factors such as cost of these medicines [6], their toxic effects [7], and reported cases of resistance development [5], their patronage and usefulness especially among people living in low-middle income countries have been limited and substituted with cheaper alternatives, mainly, herbal medicines [7]. Several studies have documented the traditional or folkloric use of herbal medicines for helminthiases [1,4,8], and scientific justifications have been widely documented for some of these medicinal plants [2,7,[9][10][11].
e name Carica papaya is almost synonymous to plantbased anthelminthic agents. Carica papaya Linn. (Caricaceae) is well known for its nutritional and medicinal values. e various parts of the plant are used in different health conditions [12]. e leaves are thought to increase breast milk and used in the treatment of tonsillitis, ulcerative stomatitis, and gingivitis [12]. e roots have been suggested to be beneficial in managing arsenic poisoning [13]. Different preparations from the leaves have found use in managing haemorrhoids, management of asthma, treatment of urinary tract infections, poultice for sores [12], and treatment of helminth infections [14]. e fruits are used to treat indigestion [15], chronic diarrhoea, ringworm infections, bleeding piles, and amoebic dysentery [14]. e stem bark, flowers, roots, and seeds have all been well documented to be used for the management of several conditions, chiefly among them are their roles in managing helminth infections [4,7,14,16].
In Ghana, a study conducted showed that 74% traditional healers used the plant for helminth infections [4]. A previous study conducted established in vitro anthelminthic activity for the leaves and the stems [16] against Pheretima posthuma. Another study established the in vitro activity of the seeds against intestinal worms, Haemonchus contortus, Oesophagostomum sp., Trichostrongylus sp., and Cooperia sp. [7]. It could be argued that due to differences in the chemical composition of the various parts of the plant, there may be observable differences in their anthelminthic activities. e current study, thus, sought to compare the activities of these parts and provide knowledge on the most effective part of the plant. e study further considered identifying which group of phytochemicals may be responsible for the activity.

Glassware and Apparatus.
Erlenmeyer's flasks, test tubes and rack, funnel, sieve, measuring cylinder, desiccator, dropper, plastic containers, petri dishes, TLC plates, chromatography columns, cotton wool, retort stand and clamp, cover plates, reagent bottles, filter paper, dropper, spatula, droppers, stirring rod, conical flask, beaker, stop watch, pencil, meter rule, and mortar and pestle were used at different stages in the study.

Collection and Preparation of Plant
Materials. e leaves, stem bark, and seeds of Carica papaya Linn. (Caricaceae) were used for the study. ese were collected on October 22, 2018, from a farm located at Krobo-Odumase in the Eastern Region of Ghana. ey were then authenticated at the Department of Pharmacognosy, Central University, School of Pharmacy, with voucher specimen number kept at the school's herbarium. e voucher specimen numbers for the samples were as follows: seeds, CARICA Seeds/2018/01; leaves, CARICA Leaves/2018/02, and stem, CARICA Stem/ 2018/03. e samples were thoroughly washed with distilled water, to get rid of unwanted foreign matter, cut into pieces, and dried separately under shade in a cool dry environment for four weeks. e dried samples were then milled into fine powder and stored in airtight plastic containers and labelled accordingly. e samples were later subjected to extraction, phytochemical analysis, anthelmintic activity investigation, and chromatography.

Test Organisms.
Adult earthworms, Pheretima posthuma (Pheritimidae), were used for the study because of their anatomical and physiological resemblance with the human intestinal roundworm parasite, Ascaris lumbricoides [2,5]. e organisms were collected from the moist soils of the biodiversity forest in Central University, Miotso in the Tema Metropolis, and washed with normal saline, to remove foreign and faecal matter. ey were then authenticated in the Department of Anatomy, School of Medicine and Health Sciences, Central University. e selected earthworms were 5 to 7 cm in length and 0.1 to 0.3 cm in width ( Figure 1).

Extraction.
For extraction, the powdered samples for each plant part were divided into two: the first in each case was extracted with ethanol (98%) while the second was extracted with a hydroalcoholic solvent (ethanol and water in the ratio 2 : 1). Plant samples were cold macerated for 72 hours, after which filtrates were concentrated in vacuo, packaged, and stored for later use (Figure 2).

Phytochemical Screening.
e freshly prepared crude extracts from the leaves (LE and LHE), stem (STE and STHE), and seeds (SE and SHE) of Carica papaya were subjected to standard qualitative phytochemical screening tests for the following secondary metabolites: tannins, alkaloids, fixed oils, reducing sugars of glycosides, and saponins according to methods described in the literature [17][18][19].

Preparation of Solutions.
1 mg/ml, 2.5 mg/ml, and 5.0 mg/ml concentrations of the crude extracts obtained ( Figure 2) were prepared by dissolving specified quantities of the extracts in normal saline. Similar concentrations were also prepared from the albendazole tablets by suspending specified quantities of the crushed tablets in normal saline.

In Vitro Anthelminthic Effects of Crude Extracts.
e method adopted was as described in literature with modifications [6,16]. Eight groups of test agents were administered to twenty two groups of adult earthworms, with each group containing three earthworms of approximately equal size. e test agents were albendazole and normal saline, serving as the positive and negative control groups, respectively, and the six crude extracts were from the leaves (LE and LTHE), seeds (SE and SHE), and stem (STE and STHE). With the exception of the negative control group (which had only one group of test organisms), all the other test agents were tested at the three concentration levels, that is, 1 mg/ml, 2.5 mg/ml, and 5.0 mg/ml (Table 1).
10 ml of each test concentration from the test agents was administered to the adult earthworms in the respective groups. Observations were made for the time taken for paralysis and death of individual worms. Paralysis was said to have occurred when the test organisms had lost mobility [20]. Similarly, death time was recorded when the test organisms were observed not to have survived, followed by fading of their body colour [20].

Bioassay-Guided Fractionation.
Guided by the outcome of the anthelminthic effects from the crude plants, SE was selected for further investigation. e SE extract was fractionated using column chromatography with silica as the stationary phase. An optimized ratio of hexane : ethyl acetate (ratio of 4 : 1) was then adopted to achieve fractions from the SE extract, which were collected in Erlenmeyer flasks and labelled accordingly. TLC analyses were carried out on the fractions obtained using the above-mentioned solvent system. e TLC chromatogram led to the bulking of some of the fractions together based on the spot patterns observed. Five fractions were then achieved after which in vitro anthelmintic testing was carried out on them, to identify the most active fraction(s).

Statistical Analysis.
e data obtained were analysed using GraphPad Prism (Prism 6 for Windows, 2012, GraphPad Software Inc., USA). e data from the evaluation of the paralysis and death times were expressed as mean-± standard error (SEM) of three earthworms in each group. Differences between means were evaluated using either the Student's t-test or analysis of variance (ANOVA) depending on the number of groups being compared. ANOVA analysis was usually followed by the Tukey post hoc test used to quantify statistical difference between specific groups. Statistical significance was established in situations with p values ≤ 0.05.

Results and Discussion
e phytochemical investigation showed the presence of alkaloids, saponins, and reducing sugars of glycosides present in all the crude extracts of Carica papaya ( Table 2). Tannins were observed only in extracts of the leaves while fixed oils on the other hand were only present in the extracts of the seeds. is observation was showed to be consistent with the observations of previous studies whichreported the presence of tannins in the leaves [21,22] and fixed oils in the seeds [23]. Some of the observations made were also not consistent with previously reported data, for example, the presence of tannins in the seeds [24] and absence of tannins in the leaves [16,25]. e differences in the chemical composition of the test samples could be theorized to be responsible for differences in their anthelminthic effects [26], as evident in the study (Table 3 and Figure 3).
From the in vitro anthelminthic investigations, it was observed that the effects of the extracts on paralysis and death times were concentration dependent; increasing the concentration of the extract from 1 mg/ml to 5 mg/ml Evidence-Based Complementary and Alternative Medicine caused significant decline in both the paralysis time (F (6,14) = 640.7, p < 0.0001) and death time (F (6,14) = 1270, p < 0.0001) recorded (Table 3). Generally, it was observed that the paralysis and death times for the crude extracts were significantly different from each other (Figure 3). Tukey's post hoc tests showed that the recorded reduction effects for all the plant extracts on paralysis and death times were significantly higher than that from albendazole (p < 0.0001). is indicated that the plant parts, seeds, stem bark, and leaves of Carica papaya, possessed significant in vitro anthelminthic effects against Pheretima posthuma.
is observation was found to be consistent with outcomes of previous studies reporting of the anthelminthic effects of seeds [7,27,28], stem barks and leaves [16], and the latex     [-] no observable immobility. Results are expressed as mean ± SEM. N � 3. Two-way ANOVA analysis, followed by Tukey's post hoc test was used to test for differences between paralysis and death times of plant extracts and albendazole (labelled as a 0.05 < p < 0.01; b 0.01 < p < 0.001; c 0.001 < p < 0.0001; d p < 0.0001) and also differences among the concentrations of each plant extract (also labelled as 1 0.05 < p < 0.01; 2 0.01 < p < 0.001; 3 0.001 < p< 0.0001; 4 p < 0.0001) using 1 mg/ml as the reference, at 95% confidence level. "ns" indicated no significant difference upon comparison of groups in all cases. Evidence-Based Complementary and Alternative Medicine from the fruit [29]. It was also showed that the effects from the plant parts investigated were higher than those of the conventional anthelminthic drug, albendazole, at the concentrations adopted (p < 0.0001). Similar observation was made in a study involving the hydroalcoholic extracts of the leaves and stem using the same model [16]. ese observations, thus, confirm and authenticate the folkloric use of the plant parts in the treatment of helminth infections [4,14]. e similar phytocomposition for the ethanolic and hydroethanolic extracts from each plant part (Table 2) was thought to contribute to the comparable activities of such extracts in the study (q = 0.8651−3.861, df = 14; Figure 3(b)). is was however not the case with their effects on death time, where it was shown that only extracts from the leaves were comparable (q = 4.392, df = 14; Figure 3(d)). It was also observed that SE and SHE extracts demonstrated the highest anthelminthic effects (Figure 3). A previous study had shown that that the seeds were comparable to thiabendazole in terms of their activity against gastrointestinal helminths in Red Sokoto goats [7]. Another study had concluded that the seeds were safe and efficacious in eliminating intestinal helminths [30]. As earlier argued, this could be as a result of their peculiar chemical composition with the presence of alkaloids, saponins, glycosides, and fixed oils. Since some of these phytoconstituents have been documented to possess anthelminthic activities [31], their presence thus may contribute to the observed activity. is observation may also confirm the relative preference of the seeds to the leaves and stem bark for traditional helminth infection management [4].
Further fractionation of the SE extract yielded SE B fraction, which also demonstrated anthelminthic effect (Table 4). It also showed that SE B demonstrated a significantly higher activity than SE, both in the reduction of paralysis time (t � 61.49, df � 2, p � 0.0003) and death time (t � 98.72, df � 2, p � 0.0001) (Figure 4). Phytochemical investigation of the SE B fraction showed the presence of fixed oils (Table 4). e fixed oils from the seeds have been showed to contain myristic, palmitic, stearic, arachidic, behenic, and unsaturated fatty acids among others [32], some of which possess anthelminthic effects against  Caenorhabditis elegans [31]. It could thus be inferred that the possible presence of these compounds may account for the high activity of the fixed oils in SE B .

Conclusions
e results obtained from this work validate the traditional or herbal practitioners' and indigenous folks' use of Carica papaya, especially the seeds as an anthelmintic agent in the treatment of helminthiasis or worm infestation.

Data Availability
e processed data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest. Evidence-Based Complementary and Alternative Medicine 7