Dro Et Al.Pdf

Research Paper

In vivo evaluation of antihelmintic potential of Napoleonaea vogelii Hook & Planch (Lecythidaceae): A traditionally used nematocidal medicinal plant in West Africa

Accepted 16th May, 2019

ABSTRACT

The need for research into alternative therapeutic agents for the control of helminth infections is of great interest for animal health. Medicinal plants play a considerably role in the discovery of new molecules. In this study, in vivo antihelmintic potential of Napoleonaea vogelii leaves was evaluated against gastrointestinal nematodes (GINs) in sheep. An 80 mg/kg single dose oral suspension of B Dro1,5*, D. Soro2,5, Dea Amon1, Mw Koné3,5 leaves ethanolic extract was administered to 3-6 months lambs aged naturally and K Kamanzi4,5 infected with GINs. Biological (bodyweight, diarrhea index), hematological 1UFR Agroforesterie, Université JEAN (Famacha, hematocrit) and parasitological (fecal eggs count reduction, reduction LOROUGNON GUEDE de Daloa, Côte d’Ivoire. of adult worms) parameters were determined. The results showed that the 2UFR Sciences Biologiques, Université ethanolic extract generated a weight gain (5.8%) in treated sheep and showed PÉLÉFORO GON COULIBALY de Korhogo, Côte d’Ivoire. moderate action on diarrhoea (diarrhoeal index = 67.3%) but had weak effect on 3UFR Sciences de la Nature, Université hematocrit as compared with the control (Untreated) group. N. vogelii was highly NANGUI ABROGOUA d'Abidjan, Côte d’Ivoire. effective against adult Oesophagostomum columbianum (94.4%) and Cooperia 4UFR Biosciences, Université Félix curticei (100%), but exhibited a marginally lower activity against Gaigeria HOUPHOUËT-BOIGNY d'Abidjan, Côte d’Ivoire. pachyscelis (61.5%) and Trichostrongylus axei (42.2%). These results indicate that 5Centre Suisse de Recherches Scientifiques en N. vogelii exhibited some potential and can be used as natural antiparasitics for the Côte d’Ivoire (CSRS) control of GINs in small ruminants.

*Corresponding author. E-mail: [email protected]. Tel: +225 09405056 / Key words: Napoleonaea vogelii, in vivo efficacy, gastro-intestinal nematodes, +225 05445166. Fax: 00225 32787570. small ruminants, West Africa.

Abbreviations: ANOVA, Analysis of variance; BW, bodyweight; °C, degree celsius; CSRS, Centre Suisse de Recherches Scientifiques en Côte d’Ivoire; %E, percentage of efficacy; et al., et alliori (and others); FECR, fecal eggs count reduction; %H, hematocrit rate; mg/kg, milligram per kilogram; μm, micrometer; GINs, gastro-intestinal nematodes; I (I1, I2, I3), diarrheal Index; NA, not applicable; M, magnification of microscope; ml, milliliter; N, north; NaCl; chloride Sodium; PASRES, programme d’Appui Stratégique à la Recherche Scientifique; SEM, standard error of mean; UFR, Unité de Formation et de Recherche; VICH, veterinary international cooperation on harmonization; WAAVP, world association for the advancement of veterinary parasitology; W, west.

INTRODUCTION

Parasites of livestock cause diseases of socio-economic severe harm and death to domestic animals (Kaminsky et importance throughout the tropics. The current financial al., 2013). and agriculture losses caused by parasites have a As in developed countries (Epe and Kaminsky, 2013), in substantial impact on farm profitability worldwide, mainly many West African countries treatment of helminth in West African poor resource countries (Roeder et al., infections remains the mainstay of worm control in grazing 2013). In Sub-Saharan countries, gastrointestinal animals and also is based on imported manufactured nematodes infections (GNIs) are responsible for heavy antihelmintics beside medicinal plants. However, cost, direct and indirect losses in livestock (Waller, 1987), due to illiteracy and unfamiliarity with imported antihelmintics, Academia Journal of Medicinal Plants; Dro et al. 147

resulting in incorrect usage in rural areas may cause “Djallonké,” from 6 to 12 months aged, were bought at the development of resistance favored by genetic features of market of Port-Bouët (Abidjan). These sheep were naturally many parasitic helminths of veterinary importance. infected by GINs from their local origin. They were reared Antihelmintic resistance poses a large threat to welfare of and fed under same conditions. farm animals (Shabaly, 2013). The constraints in the use of The animals were selected using recommendations of the conventional medicines in West Africa underline the World Association for the Advancement of Veterinary advantages of medicinal plants as an alternative treatment. Parasitology (WAAVP) and the Veterinary International Livestock producers in rural continue to use those plants as Cooperation on Harmonization (VICH) (Powers et al., 1982; dewormers, drawing upon centuries of knowledge. Coles et al., 2006). Plant based medicines used to treat gastrointestinal Sheep were distributed in three groups of four sheep for infections in earlier times have been credited with having groups I and II and five sheep for group III according to the specific antihelmintic activity (Mehlhorn et al., 2011). Most mean of Fecal Eggs Count Reduction (FECR) observed a of the West African medicinal plants screening for action week before treatment. Mac-Master method modified by against helminths have been shown to possess high in vitro Gordon and Whitlock (1939) was used for FECR. Solution of potential. However, according to Klimpel et al. (2011), chloride Sodium (NaCl, D = 1.19) was used for parasites although in vitro testing of plant extracts for antiparasitic eggs floating. efficacy provides good results, it does not always guarantee For in vivo assays, extract (80 mg/kg) and fenbendazole in vivo test systems. Therefore, suitable in vivo animal (7.5 mg/kg) were suspended in a solution composed of models provide a more accurate reflection of the Tween 80 (5%) and water (95%). Groups I and II (positive effectiveness of plant extracts. control) were treated at single oral dose of 80 and 7.5 Napoleonaea vogelii Hook & Planch (Lecythidaceae) is a mg/kg, respectively with the ethanolic extract of N. vogelii perennial shrub which is native of upper Guinean forest. leaves and fenbendazole. Group III (negative control) The geographical distribution of this plant species extends received distilled water. from Sierra Leone to Nigeria, covering all the coastal countries such as Côte d'Ivoire (Keay et al., 1964). This Evaluation of in vivo antihelmintic efficacy plant has several important medicinal properties, and is used to treat ailments such as abdominal pain, worm The efficacy of N. vogelii leaves was evaluated using fecal infections, diarrheas and stomach ache (Akah et al., 2007). egg reduction and adult worm reduction. Biological and It has exhibited in vitro antihelmintic against Haemonchus parasitological parameters such as weight gain, diarrheal contortus (Diehl et al., 2004), Schistosoma mansonii and index, rate of anemia and fecal egg count reduction (FECR) Heligmosomoides bakeri (Koné et al., 2012). No toxicity was test were recorded each three days during the three weeks reported for N. vogelii (Koné et al., 2012). (Soro et al., 2013), and the necropsy was followed after this This current study was conducted to examine the in vivo period (Coles et al., 2006). effects of N. vogelii against nematodes in sheep naturally infected with internal parasites. Bodyweight gain

Animals were weighted using anybody weighs balance and MATERIALS AND METHODS weight gain was expressed using the formula below:

Selection of the studied plant and preparation of crude Bodyweight gain (%BW) = [(BWx – BWo) / BWo] × 100 extract BWo = bodyweight before treatment; BWx = bodyweight after treatment The leaves of N. vogelii were collected in july 2010 in Azaguié (South-Eastern Côte d’Ivoire), latitudes 5°35’ and 6°15’ N and longitude 3°55’ and 4°40’ W. Leaves were Diarrheal index cleaned, dried under air-conditioning room (18 °C) and grounded to obtain powder. Crude extract was prepared Diarrheal index was based on observation of the back-train from 100 g of powder in 100 ml of ethanol 90%, under stain of animals. Feces consistency was scaled (I1 = liquid mechanical stirring (3 × 24 h). The ethanol was evaporated feces; I2 = soft feces; I3 = normal feces) using visual charter in a rotary evaporator (rotavapor) at 40C, frozen and of Cabaret (2004). The percentage of appearance of each lyophilized. values of the scale was calculated for each studied group of sheep.

Animals and parasites Hematocrit

Thirteen ewe-lambs of West African Dwarf sheep Blood was collected from jugular vein in heparinized tube Academia Journal of Medicinal Plants; Dro et al. 148

(Improvacuter ®) and centrifuged at 4000 rpm during five tested was performed with variance analysis (one-way minutes in a centrifugal machine of hematocrit (J.P. Selecta ANOVA) using SPSS 20. When ANOVA showed significant ®). The values of hematocrit were read with a reading grid difference between the three groups tested, the (Murray et al., 1977) and expressed in percentage of complementary test of the multiple comparisons of means hematocrit (% H) using the following formula below: (Turkey test) was applied to determine the level of relationship between groups (Westlake, 1971). The test % H = [(Hx – Ho) / Ho] × 100 was significant if P < 0.05. The correlation between quantitative parameters such as strongles, hematocrit and Ho = Hematocrit before treatment; Hx = Hematocrit after bodyweight was determined at P < 0.05. treatment

RESULTS Fecal egg count reduction test Effects on biological parameters During 28 days (one week pretreatment and three weeks post-treatment), fresh feces were collected from the rectum Bodyweight gain of sheep. The eggs and coccidia oocysts were counted using Mac-Master method. The efficacy of the extract was A weight gain of 5.85 ± 1.88% was recorded three weeks determined on the basis of percent reduction in the fecal post-treatment in treated group with N. vogelii. The eggs count according to the formula of Coles et al. (1992): dewormed sheep in group I had a higher average weight gain than those treated with fenbendazole (-0.3 ± 1.3% -0.3 FECR = (1-Mx / Mxo) × 100 ± 1.2%) which in turn always exceeded the untreated group (- 4.6 ± 1.5%). Statistically, N. vogelii was the most effective Mx = means of fecal eggs count after treatment; Mxo = (Table 1). However, there was no significant difference means of fecal egg count before treatment between group II (fenbendazole) and group III (negative control). Counting of adult worms during necropsy

After sacrificing animals by throat cutting, the digestive Hematocrit tract was removed and abomasum, small intestine and large intestine were separated by binding at the two ends There was a decrease of hematocrit of -1.0 ± 1.2% and - 6.2 and different junctions between organs (Graber and ± 1.5% in group I (N. vogelii) and group III (negative Perrotin, 1983; Kaufmann, 1996). The delimited control) respectively. In group II (fenbendazole), an compartments were opened longitudinally. Contents were increase of hematocrit (5.7 ± 1.3%) was recorded three collected under tape water in a bucket and passed through weeks post-treatment (Table 1). Statistically, there was a a sieve (200 μm). The residues were spilled in another significantly difference between the plant extract and bucket and three liters of water were added. The fenbendazole that was the most effective. suspension was homogenized; 200 ml were taken for a systematic identification and counting of parasites with optical microscope (M × 100). The corresponding number Diarrheal index of adult worms counted in three liters was arithmetically calculated. The percent efficacy (%E) of treatment was After administration of N. vogelii leaves extract, a stop of expressed using the formula below: diarrhea was observed in animals during the first week, with 100% of diarrheal index. However, at the end of the % E = (Mc – Mx) / Mc × 100 experiment, only 67.5% of treated animals with this extract gave normal feces. With fenbendazole, 58% of feces were Mc = mean of adults worms in group III (control negative); normal (I3) and 42% soft (I2) at three weeks after Mx = mean of adults worms in treated groups (I or II). treatment. In group III, 53% of normal feces (I3) and 47% According to Reinecke (1973, 1980) and Powers et al. of soft feces (I2) were recorded at the end of the assays (1982), efficacy of extract was classified as high (% E > 90 (Figure 1). %); moderate (%E = 80–90 %); low (% E = 60–80 %), and ineffective (% E < 60%).

Effect on fecal egg excretion Statistical analysis The extract of N. vogelii leaves was ineffective on fecal egg Comparison of means between the three groups of sheep reduction of all studied gastrointestinal parasites, with %E Academia Journal of Medicinal Plants; Dro et al. 149

Table 1: Effect of Napoleonaea vogelii leaves extract on hematocrit and bodyweight in naturally infected sheep.

Treatments Hematocrit (%) Before treatment Post-treatment Week 0 Week 1 Week 2 Week 3

Group I: N. vogelii leaves ethanolic extract (Essay) Mean ± SEM NA -1.9 ± 5.0 -2.6 ± 4.3ab -1.0 ± 1.2a Group II: Fenbendazole (positive control) Mean ± SEM NA -2.3 ± 4.5 6.7 ± 3.0b 5.7 ± 1.3b Group III: Untreated (negative control) Mean ± SEM NA -5.0 ± 0.9 -8.1 ± 4.3a -6.2 ± 1.5a P 0.43 0.002 0.000

Bodyweight gain (%) Group I: N. vogelii Mean ± SEM NA 2.3 ± 1.4b 4.9 ± 1.7b 5.9 ± 1.9b Group II: Fenbendazole (positive control) Mean ± SEM NA -0.3 ± 1.3ab -0.2 ± 1.9ab 0.3 ± 1.2a Group III: Untreated (negative control) Mean ± SEM NA -3.3 ± 1.6a -7.3 ± 2.2a -4.6 ± 1.5a P 0.061 < 0.000 < 0.000

Values with the same superscript letter are not significantly different (P < 0.05 or P < 0.001). SEM: standard error of mean, NA: not applicable.

Group I : N. Vogelii Group II : Fenbendazole Group III : Untreated

120 Week 0 Week 1 Week 2 Week 3 100 100 88 88 80 75 80 70 75 63 60 58 60 50 53 40 42 40 37 25 25 25 27 Index diarreal (%) Indexdiarreal 20 20 20 20 10 12 12

0 Index1 Index2 Index3 Index1 Index2 Index3 Index1 Index2 Index3 Index1 Index2 Index3

Figure 1: Effect of Napoleonaea vogelii leaves extract on diarrheal index in natural infected sheep. Index 1: liquid feces, Index 2: soft feces, Index 3: normal feces. Academia Journal of Medicinal Plants; Dro et al. 150

Table 2: Effect of Napoleonaea vogelii leaves on fecal eggs and oocysts excretion against gastrointestinal nematodes in naturally infected sheep.

Treatments Internal parasites Before treatment Post-treatment Week 0 Week 1 Week 2 Week 3 Strongles Group I: N. vogelii ethanolic extract (Essay) Mean ± SEM 4123 1954 ± 137b 4085 ± 1113b 4102 ± 547b FECR (%) NA 52.6 0.9 0.5 Group II: Fenbendazole (positive control) Mean ± SEM 4073 17 ± 8a 17 ± 16a 101 ± 63a FECR (%) NA 99.6 99.6 97.5 Group III: Untreated (negative control) Mean ± SEM 4118 3430 ± 270c 5020 ± 427b 6032 ± 367c FECR (%) NA 16.7 -21.9 -46.5 P value 0.00 0.00 0.00

Coccidia Group I : N. vogelii Mean ± SEM 2195 2402 ± 450 a 2093 ± 573 a 1840 ± 507a FECR (%) NA -09.4 04.6 16.2 Group II: Fenbendazole (positive control) Mean ± SEM 1902 107361 ± 49415b 142979 ± 29438c 42033 ± 8586c FECR (%) NA -5544.6 -7417.3 -2109.9 Group III: Untreated (negative control) Mean ± SEM 2008 14634 ± 2320ab 14891 ± 2884b 12610 ± 1915b FECR (%) NA -628.8 -641.6 -528.0 P value 0.00 0.00 0.00

Values with the same superscript letter are not significantly different (P < 0.05 or P < 0.001), FECR: fecal egg count reduction, SEM: standard error of mean, NA: not applicable, BW: bodyweight.

Table 3: In vivo efficacy of Napoleonaea vogelii leaves extract against adult gastrointestinal nematodes in naturally infected sheep.

Treatments Internal parasites Group III: Untreated Group I : N. vogelii Group II : Fenbendazole P Haemonchus contortus Mean ± SEM 478 ± 42b 400 ± 41b 0 ± 0a 0 PR (%) NA 16.3 100

Trichostrongylus colubriformis Mean ± SEM 592 ± 42b 506 ± 66b 26 ± 11a 0 PR (%) NA 14.3 95.6

Trichostrongylus axei Mean ± SEM 45 ± 8b 26 ± 7ab 7 ± 4a 0.009 PR (%) NA 42.2 83,3 Academia Journal of Medicinal Plants; Dro et al. 151

Strongyloïdes papillosus Mean ± SEM 121 ± 12b 124 ± 8b 0 ± 0a 0 PR (%) NA 0 100

Gaigeria pachyscelis Mean ± SEM 39 ±10b 15 ± 6b 4 ± 4a 0.01 PR (%) NA 61.5 90.4

Oesophagostomum columbianum Mean ± SEM 199 ± 7b 11 ± 4a 166 ± 12b 0 PR (%) NA 94.4 16.6

Cooperia curticei Mean ± SEM 6 ± 4 0 ± 0 0 ± 0 0.17 PR (%) NA 100 100

Trichuris globulosa Mean ± SEM 36 ± 7b 34 ± 7b 4 ± 4a 0.001 PR (%) NA 5.6 89.6

Values with the same superscript letter are not significantly different (p < 0.05 or p < 0.001), SEM: standard error of mean, NA: not applicable, PR: percentage of reduction

of 52.6 to 0.5%. Fenbendazole exhibited a intestines). access its efficacy against GINs. This plant showed percentage of egg reduction of 97.5%. Statistically, N. vogelii leaves extract showed high efficacy beneficial effects on certain biological parameters the fecal egg reduction was more important with against O. columbianum (94.4%) and C. curticei such as weight gain with an increase of 5.8% and fenbendazole than plant extract. However, as (100%). This extract’s efficacy was 61.5% against G. diarrheal index of 67.5% as compared with the compared with the negative control group, the plant pachyscelis and 42.2% on T. axei. A marginally lower control groups. According to Höglund et al. (2013), extract showed some positive effect (Table 2). The effect was observed against H. contortus (16.3%), T. such results indicate that the parasite challenge in plant extract reduced the number of coccidia at colubriformis (14.3%) and T. globulosa (5.6%). No the negative control group was sufficiently high to 16.2% while fenbendazole was totally ineffective. efficacy was recorded against S. papillosus. result in production loss. The effect on diarrhoea is Fenbendazole was effective against all the studied of interest because diarrhoea is one of the main parasites, except against O. columbianum (% E = signs of GNIs that could cause rapid death in Effect against adult worms 16.6%). Statistically, N. vogelii was more active than animals. The decrease of diarrheal index from 100% fenbendazole against O. columbianum, but the two in the first week to 67.5% in the third week can be The necropsy revealed the presence of helminths in compounds showed similar antihelmintic activities explained by the administration of a single oral sheep digestive tract, such as Haemonchus contortus against C. curticei (Table 3). dose. The plant extract was highly effective against and Trichostrongylus axei (in abomasum), C. curticei (100%) and O. columbianum (94.4%). O. Trichostronglus colubriformis, Strongyloïdes columbianum is the second prevalent nematode in papillosus, Gaigeria pachyscelis and Cooperia curticei DISCUSSION small ruminants in Côte d'Ivoire (Komoin-Oka et al., (in small intestine), Oesophagostomum 1999; Achi et al., 2003). This nodular worm is a columbianum and Trichuris globulosa (in large The in vivo antihelmintic activity of N. vogelii was considerably pathogenic parasite in sheep, goats intestine) and Tænia sp. (in large and small evaluated in naturally infected sheep in order to and cattle (Mehlhorn, 2007). In addition, Academia Journal of Medicinal Plants; Dro et al. 152

oesophagostomosis are common zoonosis found in small Akah PA, Nnaeto O, Nworu CS, Ezike AC (2007). Medicinal plants used in ruminants, cattle and primates including humans (Krief et the traditionnel treatment of peptic ulcer diseases: A case study of Napoleonaea vogelii Hook & Planch (Lecythidaceae). Res. J. Pharma. 1: al., 2008; Guillot et al., 2011). In West Africa, they are 67-74. limited only to Ghana and Togo at the moment (Guillot et Cabaret J (2004). Genetic and phenotypic diversity in benzimidazole al., 2011). N. vogelii may also have interest in controlling resistant trichostrongyle isolates from ruminants’ hosts: tools for this parasitic infection in human. control of resistance? In: Nematode Parasites Symposium: genetic diversity, virulence genes, diagnosis and control methods. C. curticei is a common parasite of the small and/or large Coles GC, Bauer C, Borgstee de FHM, Geerts S, Klei TR, Taylor MA, Waller PJ intestine that has relatively low pathogenicity. But this (1992). World Association for the Advancement of Veterinary helminth contributes to parasitic gastroenteritis in grazing Parasitology (W.A.A.V.P.) methods for the detection of antihelmintic small ruminants (Roeber et al., 2013) with main signs such resistance in nematodes of veterinary importance. Vet. Parasitol. 44(1- 2): 35-44. as anorexia and loss of bodyweight (Mehlhorn, 2011). So, Coles GC, Jackson F, Pomroy WE, Prichard RK, von Samson-Himmelstjerna efficient treatments with the extract of N. vogelii against G, Silvestre A, Taylor MA, Vercruysse J (2006). The detection of these parasites are of interest in reducing the negative antihelmintic resistance in nematodes of veterinary importance. Vet. effects of these symptoms. Parasitol. 31(3-4): 167-185. Diehl MS, Kamanzi Atindehou K, Tere H, Betschart B (2004). Prospect of Disappointingly, N. vogelii leaves was ineffective, in vivo, antihelmintic plants in the Ivory Coast using ethnobotanical criteria. 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