Extracts of carica papaya L. and capsicum annuum L. showed comparable efficacy to piperazine citrate and levamisole hydrochloride in treatment of poultry helminths

doi:10.21203/rs.3.rs-5436954/v1

Background

In rural smallholder poultry production systems, commercially available anthelmintic drugs are generally expensive, and in some instances ineffective because of resistance developed against. We report on the phytochemical properties and efficacy of crude extracts of carcia papaya and capsicum annuum against natural helminth infections of chicken in …….. District, Eastern ………..

Methods

An experiment was set to evaluate efficacy of crude extracts of C. papaya and C. annuum against natural poultry helminths infections. Commercially available formulations of levamisole and piperazine were used as control treatments. Faecal egg count reduction (FECR) tests were used to measure efficacy of the treatments.

Results

On gas chromatograph mass spectrometry (GC-MS) analysis of CPLa showed sterols (13%), Vitamin C (42%), Triterpenoids (6%). CPLe contained pyranones (20.3%), phenolics (3.1%), glycosides (2.2%), diterpenoids (4.9%), lipids (45.04%), triterpenoids (3.5%) and steroids (1.4%). Gas Chromatography-mass Spectrometry (GC-MS) analysis of CAFa gave lipids (45.04%), alkanes (27.7%) and alkaloids (8.2%). CAFe showed glycosides (3.61%), lipids (50.16%), pyranones (3.55%) and alkaloids (22.73%). In the in-vitro assays; 0.08g/ml of each of the extracts had immobilized more than 50% of adult A. galli after five hours. The ranking of the in-vivo average FECR was Levamisole hydrochloride > CPLa > CAFa > CAFe > CPLe > piperazine citrate with the percentage reductions of 98.67 ± 2.309, 97.67 ± 2.517, 79.67 ± 1.528, 76.33 ± 1.528, 54.00 ± 2.00, 35.67 ± 2.082 respectively.

Conclusion

The GC-MS analysis of the analysed plants shows presesnce of terpenoids, phenolics and alkaloids which are known for anthelmintic action. All the extracts caused higher FECR than piperazine. The presence of Vitamin C in CPLa resulted in it being as good as levamisole. Combinations of anthelmintics with Vitamin C are recommended and toxicological studies before recommendation of these alternatives.

Capsicum annuum

Carica papaya

Frequency of citation

Gas Chromatography

Mass spectrometry

Worms

Vitamin C

Medicinal plants are widely used in farming communities across Africa and Asia for the treatment and control of poultry helminth infections [1–4]. The use of natural remedies is regarded an indigenous technical knowledge in communities [5]. As such, not much scientific research is dedicated to exploring and standardizing the use of natural products in treatment and/or control of animal diseases [6]). Other than for cultural reasons, animal keepers, especially in developing countries often resort to use of natural products because commercially available drugs are considered very expensive [7]. Conventional commercial preparations may also require administration by a qualified technical person such as a veterinarian, actually seen as an additional cost to the poultry farmer [8].The practice is becoming popular because of the need of decreasing residues of synthetic drugs and the demand for organic food products [9]. Additionally, there are reports of widespread helminth resistance against common commercial anthelmintic products, hence the need for suitable alternatives [10]. Furthermore, there is an increasing campaign to promote consumption of residue-free organic foods, including animal products without anthelminthic residues [11]. Therefore, natural remedies, including safe and efficacious medicinal plants is today seen as a viable alternative to conventional anthelmintic drugs. The use of EVP is getting popular in the different parts of the world. Use of EVP is becoming popular even in the first world like UK [12] and in rural S. Africa [13]. India has a rich ethnoveterinary base [14] while China has vast ethnoveterinary experiences which have been improved overtime [15]. In Uganda, at least 80% of farmers use medicinal plants in the treatment and control of poultry diseases, including helminth parasites [16]. The freshly collected plant material is usually crushed into a paste, mixed in water and orally administered [13]. The range of plants used and the practices in application are diverse. Specifically, capsicum and papaya plants are reportedly used widely in ethnoveterinary practice, including for poultry helminth control [17].

Helminths cause production losses and predispose chicken to microbial infections [18–21]. Helminths lower chicken vaccine responses and can affect the general immune system of chicken. However in mild and subclinical cases the signs of helminthiasis may not be observed [22]. Simultaneous presence of Heterakis gallinarum and Histomonas meleagridis in chicken increases Th1 cells and decreases splenic CD4 + cells [23]. Some helminths affect the gastro-intestinal tract resulting into poor digestion and reduced absorption of food [24].Worm migrations lead to mechanical damages of various organs. Helminths cause stress to chicken [25]. Clinical helminths infestations causes unthriftness, diarrhoea, innappetence and stuntedness among chicken [26]. Syngamus trachea causes respiratory distress. Heterakis gallinarum hosts Histomonas meleagridis which causes histomoniasis (typhllo-hepatitis) [27]. Ascaridia galli are very large nematodes that lead to intestinal obstructions especially in chicks. Ascaridia galli develops various associations with bacteria resulting into more tissue damages and mortalities to chicken [28–29]. Phenothiazine and piperazine combinations which were effective on Heterakis gallinarum, Ascaridia galli and tapeworms were banned by the Food and Drug Agency (FDA) of the United States [30].

Carica papaya L. tree is a giant tropical herb with a semi-woody usually single stemmed growing to about 10m high [31]. The leaves are about 60cm wide with deeply palmated lobes. The leaves are attached to long hollow stalks [32]. Capsicum annum L. are tropical perennial shrubs of the family Solanaceae. Although the species refers to a variety of peppers, this work refers to Capsicum annuum var. annuum with the smallest fruits. The fruits are red and taper gradually being pointed at the end. There is limited published information on phytochemical composition and effectiveness of these products. We thus, present results of phytochemical composition, in vitro and in vivo experiments to test the efficacy of Capsicum annuum L. and Carica papaya L. extracts against poultry helminths.

2.1 Description of study area

The study was in ………………………. (Fig. 1). ………. district is located at Latitude 1⁰42^I 47.4516^IIN and Longitude 33⁰36^I 22.986^IIE. It is one of the areas with high number of households keeping chicken in Eastern ……………. [33].

Figure 1. Map showing the sub-counties of ………………………………., adopted from (……………for anonymity).

2.2 Study design

This was a controlled experiment designed initially to test in vitro efficacy of crude extracts of Carica papaya L. and capsicum annuum L. using piperazine citrate as positive control. The extracts were further tested in live chicken randomly assigned to different experimental treatments including levamisole hydrochloride and piperazine citrate. The study followed a survey on plants used against chicken helminths.

2.3 Description and care of experimental chicken

A total of one hundred (100) one day indigenous chicken were raised on free range with mother hens and offered night shelters. The chicks scavenged with the mother hens and no drugs were administered except the Newcastle virus vaccination. They were purposively left to acquire helminths naturally during feeding. They were selected for experiments in the 7th week.

2.4 Preparation of acetone and ethanolic extracts

Fresh leaves of Carica papaya (pawpaw) and ripened fruits of Capsicum annuum (bird’s eye chili) were collected from homesteads in ………… between May-June, 2024). The collected plant materials were washed with tap water to remove observable debris and air dried in a shade on the farm of the Faculty of Agriculture and Animal Sciences, ………. University for two weeks. Using a coffee grinder (silver crest®) model SC-1880, from Guangzhou China; the dried plant materials were ground into fine powder.Extracts were obtained Using the conventional soxhlet method [34] with minor modifications. Ten grams of ground plant material with 5g of pumice stone were placed in a cellulose thimble plugged with cotton. Extraction was performed using a solid to liquid ratio of 1 to 12 (g/ml) for 8 hours. The extraction was done in duplicate using analytical grade ethanol and acetone as solvents. The extract was concentrated under vacuum at 40^oC and kept at 4^oC pending use.

2.5 Phytochemical and GC_MS analysis of plant extracts

Phytochemical analysis of acetone extracts was done according to the methods of Harborne [35]. Phytochemical analysis of ethanol extracts was done according to the methods of Ejikeme et al. [36], Rao et al. [37], Chauke et al. [38], Sorescu et al.[39], Sankhalkar &Verneka, [40]. For GC-MS analysis, dried extract was dissolved in 50µL moxifloxacin (Mox) and held at 37^oC for 90 minutes. The derivatization was initiated by adding 50µL N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA) and 1% tert-Butyldimethylsilyl (TBDMS) followed by incubation at 55^oC for 60 minutes. After centrifugation at 13000 rpm for 10min, the supernatant was collected and analysed by GC-MS.

GC-MS: (GC-MS- agilent), model 7000D triple quadruplets equipped with a split injector (Split ratio 1:0) was used. The injection temperature setting was at 250°C and the injected volume was 1µL. The column (ZB-5SMi, 30 m × 0.25 mm × 0.25 µm) was used. The column temperature program was employed in which the initial temperature was 80°C, held for 20 min, followed by a temperature increase at 5°C min/min to 180°C, then held for another 5 min to 250^oC, and 15 minutes to 310^oC. Helium was employed as the carrier gas at an average linear velocity of 44 .5cm/sec, prime pressure of 500–900. The flow control mode had pressure at 99.8kPa, total flow (50mL/min), column flow (1.46mL/min), linear velocity (44.5 cm/sec), and purge flow (5.0 mL/min). Data were processed on GC-MS and compounds were identified by comparison with the National Institute of Standards and Technology (NIST) in the GC-MS library.

2.6 In-vitro susceptibility tests

Naturally infected local breed chicks of 7–8 weeks described in section 2.3 above were used as the source of helminths. The birds were sacrificed and the Ascaridia galli collected from the intestines (Fig. 7). The Ascaridia galli from intestines of chicken were washed in PBS (0.01M PBS, 0.138M NaCl, 0.0027M KCl), counted and used for in-vitro efficacy assays immediately. The acetone and ethanol extracts were diluted to strengths of 0.32, 0.16, 0.08, 0.04, 0.02 g/ml using (2% DMSO in PBS), ten millilitres (10ml) of the extracts were poured in the petri dishes and 10 worms were added to each petri dish. 0.025g/ml piperazine citrate (interchemie-Holland) was used as a positive control while 2% DMSO in PBS was used a negative control. The worms were observed for skin damages, motility inhibition and deaths in intervals of 30minutes for 300 minutes.

2.7 In-vivo efficacy experiment

The chicken for the experiment were selected from those described in section 2.3 above. On the day of recruitment, each chicken was kept in isolation for between 30-60minutes or until it voided a faecal dropping. The faecal dropping was then subjected to helminth egg identification and counting as described by Glennon, [41]. Chicken that had stool with helminth egg counts above 200epg were selected for experimental treatments. The selected chicken each weighing about 300-350g were put in cages and kept inside a well ventilated poultry house (Fig. 2). At least three (03) experimental chicken were kept in each cage (0.16m²). Commercial growers mash (Nuvita®) was fed with each bird receiving about 55g of the mash daily. Tap water was given ad libitum. The birds were allowed to acclimatise in this condition for seven (07) days prior to experimental treatments. Each chicken was forcefully with a syringe administered with 3ml (0.48g) per os of the extract for the extracts groups, levamisole was given at 25mg/kg and Piperazine at 35mg/kg. The concentration of the plant extracts were determined from previous in vitro experiments as double the lowest concentration that inhibited motility of the highest number of A. galli worms. The lowest in-vitro concentration that inhibited motility of more than half of the mature A.galli was 0.08g/ml of the extract for most extracts, it was doubled to 0.16g/ml in-vivo concentration and the birds received 0.48g for each day. The treatment was repeated on the second day.

A week after treatments, faecal samples per treatment were collected for egg count per gram of faeces determination from the Central diagnostic laboratory, College of Veterinary Medicine, ………. University. Egg counts were determined by modified Mcmaster technique [41].

2.8 Statistical Analysis

Phytochemical percentage compositions were tabulated in Ms-excel and presented in tables. In-vitro data of number of non-motile Ascaridia galli (number of worms that couldn’t move or turn their bodies when tapped with a small metal rod) were entered in MS-excel in triplicates and transferred to SPSS version 26. One way ANOVA was used to determine any significant difference between the means of number of non-motile Ascaridia galli per treatment. Tukey’s HSD was used to determine the specific treatment groups that were different from each other. The in-vivo data of ECG was entered in MS-excel in triplicates and transferred to SPSS version 26. One way ANOVA was used to determine any significant difference between the means of ECG per treatment. Tukey’s HSD was used to determine the specific treatment groups that differed from each other. The differences were considered significant when p ≤ 0.05.

2.9 Ethical considerations

An institutional ethical review certificate was acquired from the school of Veterinary medicine and Animal Resources, College of Veterinary Medicine and Biosecurity, ………… University.

The permission to collect plant materials which were both wild and planted was granted by the ……… National Council of Science and Technology (……..) under permission number- A220ES. The guidelines that were given by Institution Review Board and ……... were followed and no other licenses were required.

3.1. Qualitative Phytochemical analysis

The results are detailed in Table 1 below;

Table 1

Qualitative Phytochemical analysis of *Carica papaya* L. and *Capsicum annuum* L.
Compound	Carica papaya L. extract		Capsicum annuum L,
Compound	(CPLe)	(CPLa)	(CAFe)	(CAFa)
Saponins	+	-	-	-
Tannins	-	-	-	-
Reducing compounds	+	-	+	-
Alkaloid salts	+	+	+	+
Anthocyanosides	-	-	+	-
Anthraconosides	-	-	+	-
Coumarins	+	+	+	+
Flavonosides	+	+	+	+
Steroid glycosides	+	+	+	+

Capsicum annuum L. & Carica papaya L. showed presence of alkaloid salts, coumarins, flavonosides and steroid glycosides. Only ethanoic extracts of Capsicum annuum L. showed presence of anthrocyanosides and anthraconosides. Only ethanol extracts of Carica papaya L. showed presence of saponins. None of the extracts showed presence of tannins.

3.2. GC-MS profile of Carica papaya L. and Capsicum annuum L. extract

(CPLa) yielded sterols (13%), vitamin C (42%) and triterpenoids (6%) (Table.2 & Figure. 3). (CPLe) yielded pyranones (20.3%), phenolics (3.1%), glycosides (2.2%), diterpenoids (4.9%), sterols (33%), triterpenoids (3.5%) and steroids (1.4%) (Table.3 & Figure. 4). (CAFa) yielded sterols (45.04%), alkanes (27.7%) and alkaloids (8.2%) (Table.4 & Figure. 5). (CAFe) yielded glycosides (3.61%), sterols (50.16%), pyranones (3.55%) and alkaloids (22.73%) (Table.5 & Fig. 6).

Table 2

GC-MS profile of *Carica papaya* L. leaves acetone extract (CPLa)
Retention time	Compound Name (AP)/ Group of compound	CAS#	Formula	Component area	Match factor	Estimated conc. (%)
4.1974	2H-Benzo[f]oxirenol[2,3-E]benzofuran-8(9H)-one,9-[[[2-(dimethylamino)ethyl]amino]methyl]octahydro-2,5a-dimethyl-	1000316-31-0	C₁₉H₃₂N₂O₃	30902350.6	57	31
16.7910	9-Hexadecenoic acid, 9-octadecenyl ester, (z,z)- / (sterols)	22393-98-2	C₃₄H₆₄O₂	7707685.4	63.3	7.7
20.2233	1-(+)-Ascorbic acid 2,6-dihexadecanoate / (vitamin C)	28474-90-0	C₃₈H₆₈O₈	42045756.6	68.5	42
25.3152	2-Butenoic acid, 2-methyl-, 2-(acetyloxy)-1,1a,2,3,4,6,7,10,11,11a-decahydro-7,10-dihydroxy-1,1,36,9-pentamethyl-4a,7a-epoxy-5H-cyclopenta[a]cycloundecen-11-yl ester, [1aR-[1aR,2R,3S,4aR,6S,7S,7aS,8E,10R,11R(E),11aS]]- /(sterols)	51906-13-9	C₂₇H₃₈O₈	5309888.5	61.2	5.3
29.0563	D:A-Friedooleanan-3-ol,(3,alpha.)- / (Triterpenoids)	5085-72-3	C₃₀H₅₂O	6262628.8	51.2	6

(CPLa) yielded 13% sterols and 6% triterpenoids which are some of the compounds with known anthelmintic action.

Table 3

GC-MS profile of *Carica papaya* L. leaves ethanolic extract (CPLe)
Retention time	Compound Name (EP)/ Group of compound	CAS#	Formula	Component area	Match factor	Estimated conc. (%)
4.4683	Benzene,1,2- dichloro-	95-50-1	C₆H₄Cl₂	310372634.1	77.4	1.5
4.8495	Benzyl alcohol	100-51-6	C₇H₈O	656899632	74.3	3.1
5.5951	4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl- / (Pyranones)	28564-83-2	C₆H₈O₄	4244572882.5	79.7	20.3
7.0002	Phenol, 5-ethenyl-2methoxy- / (Phenolics)	621-58-9	C₉H₁₀O₂	652974980.4	81.6	3.1
8.4054	Ethyl beta-d-riboside / (glycosides)	1000126-95-4	C₇H₁₄O₅	470071693.6	70.8	2.2
16.7973	Neophytadiene /(diterpenoids)	504-96-1	C₂₀H₃₈	341714834.0	87.2	1.6
20.6459	n-Hexadecanoic acid / (sterols)	57-10-3	C₁₆H₃₂O₂	1352846816.3	64.9	6.5
21.1578	Hexadecanoic acid, ethyl ester / (sterols)	628-97-7	C₁₈H₃₆O₂	242270004.7	83.5	1.2
24.5382	Phytol /(diterpenoids)	150-86-7	C₂₀H₄₀O	699650422.6	88.3	3.3
25.6724	9,12,15-Octadecatrienoic acid (Z,Z,Z)- / (sterols)	463-40-1	C₁₈H₃₀O₂	4364890955.8	87.4	20.8
26.4739	Octadecanoic acid / (sterols)	57-11-4	C₁₈H₃₆O₂	639975153.6	84.3	3.1
36.3320	Supraene /(Triterpenoids)	7683-64-9	C₃₀H₅₀	731801424.0	84.5	3.5
43.8826	beta.-Sitosterol / (Steroids)	83-46-5	C₂₉H₅₀O	284103679.4	81.2	1.4

(CPLe) yielded 25.3% sterols, 8.4% terpenoids and 3.1% phenolics which are some of the compounds with known anthelmintic action

Table 4

GC-MS profile of *Capsicum annuum* L. fruits acetone extract (CAFa)
Retention time	Compound Name (AR)/ Group of compound	CAS#	Formula	Component area	Match factor	Estimated conc. (%)
25.8299	9,12-Octadecadienoic acid (Z,Z)- /(sterols)	60-33-3	C₁₈H₃₂O₂	1344761417.7	88.4	9.91
26.0223	9,12,15-Octadecatrienoic acid, (Z,Z,Z)- /(sterols)	463-40-1	C₁₈H₃₀O₂	275995441.8	60.1	2.03
26.4819	Pentadecanoic acid /(sterols)	1002-84-2	C₁₅H₃₀O₂	546092918.2	83.6	4.02
32.5972	Heptacosane /(alkanes)	593-49-7	C₂₇H₅₆	1745180778	81.2	12.86
33.4436	Capsaicin /(Alkaloid)	404-86-4	C₁₈H₂₇NO₃	802053360.9	84.9	5.91
33.6958	Dihydocapsaicin /(Alkaloids)	19408-84-5	C₁₈H₂₉NO₃	311451357.4	72.5	2.29
34.9720	Heneicosane /(alkane)	629-94-7	C₂₁H₄₄	748932682.8	84.2	5.52
36.3398	Squalene /(sterols)	111-02-4	C₃₀H₅₀	3032372293.2	85.0	22.34
36.8537	1-Heptacosanol /(sterol-alcohol)	2004-39-9	C₂₇H₅₆O	384232719.6	85.3	2.83
38.1879	Hentriacontane / (alkane)	630-04-6	C₃₁H₆₄	1264907160	76.5	9.32
39.3970	1-Heptacosanol /(sterols)	2004-39-9	C₂₇H₅₆O	252395509.5	83.7	1.86
41.2582	Tetratetracontane /(sterols)	7098-22-8	C₄₄H₉₀	278416640.1	71.4	2.05

(CAFa) yielded 45.04% sterols and 8.2% alkaloids which are some of the compounds with known anthelmintic action

Table 5

GC-MS profile of *Capsicum annuum* L. ethanolic extract (CAFe)
Retention time	Compound Name (ER)/ Group of compound	CAS#	Formula	Component area	Match factor	Estimated conc. (%)
6.2338	5-Hydroxymethylfurfural /(glycosides)	67-47-0	C₆H₆O₃	1039557851.9	81.7	3.61
16.1489	Oleic acid /(sterol)	112-80-1	C₁₈H₃₄O₂	1263336730	68.9	4.39
16.6178	Pentadecanoic acid /(sterol)	1002-84-2	C₁₅H₃₀O₂	806683607.3	86.5	2.80
18.8356	Palmitoleic acid /(sterol)	373-49-9	C₁₆H₃₀O₂	1194629505	84.0	4.15
19.2672	n-Hexadecanoic acid /(sterol)	57-10-3	C₁₆H₃₂O₂	1090877057	73.3	3.79
21.6884	Cic-10-Heptadecenoic acid /(sterol)	29743-97-3	C₁₇H₃₂O₂	602677070.9	81.7	2.09
26.0962	Ethanol, 2-(9,12-octadecadienyloxy)-(Z,Z)-	17367-08-7	C₂₀H₃₈O₂	1539700382	80.6	5.35
26.3316	2H-Pyran-2-one, tetrahydro-6-tridecyl- / (pyranones)	1227-51-6	C₁₈H₃₄O₂	1021059290.5	55.2	3.55
26.4343	9,12,15-Octadecatrienoic acid, (Z,Z,Z)- /(sterol)	463-40-1	C₁₈H₃₀O₂	2107179713.2	67.2	7.32
26.8005	Octadecanoic acid /(sterol)	57-11-4	C₁₈H₃₆O₂	942150689.0	83.3	3.27
28.4168	Arachidamide, N-methyl- /(sterol)	1000420-44-0	C₂₁H₄₃NO	6433662652.3	64.0	22.35
31.4010	Capsaicin /(Alkaloid)	404-86-4	C₁₈H₂₇NO₃	4834637352	65.6	16.80
33.9070	Dihydrocapsaicin /(Alkaloid)	19408-84-5	C₁₈H₂₉NO₃	1706326041	78.9	5.93

(CAFe) yielded 50.09% sterols and 22.73% alkaloids which are some of the compounds with known anthelmintic action

3.3. In-vitro Efficacy

Capsicum annuum L. extracts (CAFa & CAFe)

The extracts took long to act as compared to piperazine citrate but showed anthelmintic activity. The extracts didn’t cause any observable lesions on the skin whatsoever. Four to five hours was needed for the extracts to make Ascaridia galli immotile. A concentration of 0.08g/ ml made over 50% of the mature A.galli immotile after 5 hours while 0.32g/mL made over 80% mature A.galli immotile, Table 6.

Carica papaya L. extracts

CPLe acted on A. galli faster than Capsicum annuum L. fruit extracts. After 240 min 0.32g/ml CPLe made 75% of adult A. galli immotile. After 270 min, 0.08g/ml had made 50% of adult A.galli immotile and by 300min, 0.08g/ml had made 65% of the adult A.galli.immotile, Table 6.

CPLa by 240min, 0.08g/ml had made 55% of adult A.galli immotile and 0.32g/ml had made 100% of adult A.galli immotile. By 300min, 0.08g/ml had made 70% of adult A.galli immotile, Table 6.

Table 6

*In-vitro* efficacy tests of the extracts, positive control (Piperazine citrate) and negative control (PBS)
Treatment	Concentration g/mL	60min	90min	120min	150min	180min	210min	240min	270min	300min
CAFe	0.32	0.0 ± 0.0	0.00 ± 0.000	0.00 ± 0.000	1.33 ± 0.577	3.67 ± 0.577	4.67 ± 0.577	5.33 ± 0.577	7.67 ± 0.577	8.67 ± 0.577
	0.16	0.0 ± 0.0	0.00 ± 0.000	0.00 ± 0.000	0.33 ± 0.577	3.00 ± 1.000	3.67 ± 0.577	4.67 ± 0.577	5.67 ± 0.577	7.33 ± 0.577
	0.08	0.0 ± 0.0	0.00 ± 0.000	0.00 ± 0.000	0.00 ± 0.000	2.67 ± 0.577	3.00 ± 1.000	3.33 ± 1.528	3.67 ± 1.155	5.33 ± 0.577
	0.04	0.0 ± 0.0	0.00 ± 0.000	0.00 ± 0.000	0.00 ± 0.000	1.33 ± 0.577	1.67 ± 0.577	1.67 ± 0.577	2.67 ± 0.577	4.33 ± 0.577
	0.02	0.0 ± 0.0	0.00 ± 0.000	0.00 ± 0.000	0.00 ± 0.000	0.00 ± 0.000	0.33 ± 0.577	1.00 ± 0.000	1.33 ± 0.577	2.67 ± 0.577
CAFa	0.32	0.0 ± 0.0	1.33 ± 1.155	2.33 ± 1.155	5.33 ± 0.577	6.33 ± 0.577	7.33 ± 0.577	8.33 ± 0.577	9.00 ± 0.000	9.67 ± 0.577
	0.16	0.0 ± 0.0	0.33 ± 0.577	0.67 ± 1.155	4.00 ± 1.000	4.33 ± 0.577	5.67 ± 1.155	6.33 ± 0.577	6.67 ± 1.155	9.00 ± 1.000
	0.08	0.0 ± 0.0	0.00 ± 0.000	0.33 ± 0.577	2.67 ± 1.155	3.00 ± 1.000	4.67 ± 0.577	5.00 ± 1.000	5.33 ± 0.577	6.67 ± 0.577
	0.04	0.0 ± 0.0	0.00 ± 0.000	0.00 ± 0.000	0.67 ± 1.155	1.67 ± 1.528	3.33 ± 0.577	4.00 ± 1.000	4.33 ± 0.577	5.00 ± 1.000
	0.02	0.0 ± 0.0	0.00 ± 0.000	0.00 ± 0.000	0.00 ± 0.000	0.00 ± 0.000	0.67 ± 0.577	1.00 ± 0.000	1.33 ± 0.577	1.67 ± 0.577
CPLe	0.32	0.0 ± 0.0	0.00 ± 0.000	1.67 ± 1.155	2.67 ± 1.155	5.00 ± 1.000	7.00 ± 0.000	7.67 ± 0.577	8.67 ± 0.577	9.33 ± 0.577
	0.16	0.0 ± 0.0	0.00 ± 0.000	1.00 ± 1.000	2.33 ± 1.528	4.67 ± 0.577	5.33 ± 0.577	6.33 ± 0.577	6.67 ± 0.577	7.33 ± 0.577
	0.08	0.0 ± 0.0	0.00 ± 0.000	0.33 ± 0.577	0.67 ± 0.577	2.33 ± 1.155	3.67 ± 0.577	4.33 ± 0.577	5.33 ± 0.577	7.00 ± 1.000
	0.04	0.0 ± 0.0	0.00 ± 0.000	0.00 ± 0.000	0.67 ± 0.577	2.33 ± 1.155	2.67 ± 1.528	3.67 ± 1.155	4.00 ± 1.000	5.33 ± 0.577
	0.02	0.0 ± 0.0	0.00 ± 0.000	0.00 ± 0.000	0.00 ± 0.000	0.33 ± 0.577	0.67 ± 0.577	2.00 ± 1.000	2.67 ± 0.577	3.67 ± 0.577
CPLa	0.32	0.0 ± 0.0	0.00 ± 0.000	3.67 ± 0.577	5.00 ± 1.000	6.00 ± 0.000	9.33 ± 0.577	10 ± 0.000	10.0 ± 0.000	10.0 ± 0.000
	0.16	0.0 ± 0.0	0.00 ± 0.000	1.67 ± 0.577	4.33 ± 0.577	5.33 ± 0.577	5.67 ± 0.577	7.33 ± 0.577	8.67 ± 0.577	8.67 ± 0.577
	0.08	0.0 ± 0.0	0.00 ± 0.000	1.33 ± 0.577	1.67 ± 1.155	3.67 ± 0.577	4.67 ± 0.577	5.67 ± 0.577	7.33 ± 0.577	7.33 ± 0.577
	0.04	0.0 ± 0.0	0.00 ± 0.000	0.00 ± 0.000	0.33 ± 0.577	2.67 ± 0.577	3.33 ± 0.577	4.33 ± 0.577	5.67 ± 0.577	5.67 ± 0.577
	0.02	0.0 ± 0.0	0.00 ± 0.000	0.00 ± 0.000	0.00 ± 0.000	0.33 ± 0.577	1.00 ± 1.000	1.33 ± 1.155	2.33 ± 1.155	2.67 ± 0.577
Pip-citrate	0.025	1.0 ± 1.0	3.00 ± 1.000	8.33 ± 1.528	9.33 ± 0.577	10.0 ± 0.000	10.0 ± 0.000	10.0 ± 0.000	10.0 ± 0.000	10.0 ± 0.000
PBS		0.0 ± 0.0	0.00 ± 0.000	0.00 ± 0.000	0.00 ± 0.000	0.00 ± 0.000	0.00 ± 0.000	0.00 ± 0.000	0.00 ± 0.000	0.00 ± 0.000

The mean number of immobilized A. galli for each concentration after the specified number of minutes, (±) standard deviation

3.4. In-vivo Efficacy

All extracts were significantly more effective compared to PBS (p = 0.000) and CPLa was not significantly different from levamisole (Table 7–8)

$$\:\%\:FEC\:reduction\:test\:\left(FECR\right)=\frac{100\left(FEC\:control-FEC\:treated\right)}{FEC\:control\:}$$

Table 7

*In-vivo* Efficacy tests of the extracts, Piperazine and Levamisole
No.	Code	ECG₁	ECG₂	ECG₃	FECR₁ (%)	FECR₂ (%)	FECR₃ (%)	Mean FECR (%)
1	CAFa	2900	2805	2493	80	78	81	79.67 ± 1.528
2	CAFe	3250	3188	2886	76	75	78	76.33 ± 1.528
3	CPLa	0	638	262	100	95	98	97.67 ± 2.517
4	CPLe	6100	5865	6298	56	54	52	54.00 ± 2.000
5	PiP	9000	7905	8659	35	38	34	35.67 ± 2.082
6	Lev	0	510	0	100	96	100	98.67 ± 2.309
7	PBS	13800	12750	13120	0	0	0	0.00 ± 0.000

Egg counts per gram of faeces (ecg), faecal egg count reductions (FECR) and Mean FECR. Although CAFa& CAFe were effective but CPLa was the most effective, almost as good as levamisole.

Table 8. Multiple comparisons of In-vivo Efficacy tests of the extracts, Piperazine and Levamisole

Dependent Variable: FECR
Tukey HSD
(I) Treatment	(J) Treatment	Mean Difference (I-J)	Std. Error	Sig.	95% Confidence Interval
					Lower Bound	Upper Bound

PBS	CPLa	-97.67^*	1.533	.000	-102.90	-92.43
	CAFa	-79.67^*	1.533	.000	-84.90	-74.43
	CPLe	-54.00^*	1.533	.000	-59.23	-48.77
	CAFe	-76.33^*	1.533	.000	-81.57	-71.10
	Lev	-98.67^*	1.533	.000	-103.90	-93.43
	Pip	-35.67^*	1.533	.000	-40.90	-30.43
Pip	CPLa	-62.00^*	1.533	.000	-67.23	-56.77
	CAFa	-44.00^*	1.533	.000	-49.23	-38.77
	CPLe	-18.33^*	1.533	.000	-23.57	-13.10
	CAFe	-40.67^*	1.533	.000	-45.90	-35.43
	Lev	-63.00^*	1.533	.000	-68.23	-57.77
	PBS	35.67^*	1.533	.000	30.43	40.90
Lev	CPLa	1.00	1.533	.993	-4.23	6.23
	CAFa	19.00^*	1.533	.000	13.77	24.23
	CPLe	44.67^*	1.533	.000	39.43	49.90
	CAFe	22.33^*	1.533	.000	17.10	27.57
	PBS	98.67^*	1.533	.000	93.43	103.90
	Pip	63.00^*	1.533	.000	57.77	68.23

4.1 Qualitative Phytochemical analysis of Carica papaya L. and Capsicum annuum L.

The realised phytochemicals were those commonly known to have anthelmintic activity, using the plants as anthelmintics is contextual. Saponins have been reported to have anthelmintic effects [42–43]. Alkaloids are also reported to have action against helminths [44–45]. Coumarins have inhibitory effects against helminths [46–47]. Some flavonoids are effective against helminths [48–49]. Some steroids are effective against helminths [50].

4.2 Quantitative Phytochemical analysis of Carica papaya L. and Capsicum annuum L.

Ethanoic extracts (CPLe and CAFe) showed more compounds than acetone extracts (CPLa and CAFa). CPLa had the best in-vivo action, this opportunity can be utilised at industrial level when scaling up production. Steroids are said to have anthelmintic effects [5] but the action of specific lipids on helminths are not fully known. Vitamin C doesn’t have particular action against helminths but it improves the response of hosts to helminths infections [52]. Triterpenoids have known effects on helminths [53–54], however the specific effect of D: A-Friedooleanan-3-ol, (3, alpha.)-and Supraene against helminths requires further investigation. The action of pyranones against helminths are not known and requires further investigation. Phenolics have known action against helminths [55–56], however the particular effect of Phenol, 5-ethenyl-2methoxy- on helminths is not known. Particular glycosides especially flavonal glycosides have anthelmintic action [57] however the action of all other types of glycosides against helminths require further research. Diterpenoids have known action against helminths [58]. Phytol is known to be effective against helminths [59], but the effect of Neophytadiene on helminths requires further research. The action of alkanes against helminths are not known and require further research. Alkaloids have known action against helminths [44, 60]; Capsaicin and Dihydocapsaicin are known to be effective against helminths especially due to their pungency action [61].

4.3. In-vitro Efficacy tests of the extracts

No deaths were observed nor any observable lesions on the A. galli dermis, however inhibited motility was observed with no change of colour of worms. The extracts were significantly effective compared to PBS but acted slowly compared to piperazine citrate. The action of Carica papaya L. extracts is in agreement with the findings of Nghonjuyi et al. [62] and Sugiharto, [63]. The findings regarding Capsicum annuum L. extracts are in agreement with Gentiles et al. [64] who reported the high anthelmintic potency of Capsicum annuum var. Longum.

No major difference in the action for CAFa & CAFe because they had almost the same composition of the known phytochemical compounds.

The CPLa was faster than CPLe. It was only CPLa that contained vitamin C. All Carica papaya L. extracts had terpenoids whose action against nematodes is said to be boosted by vitamin C. The findings are in agreement with Sen et al. [65] who found 100% in-vitro effect against A. galli at even 20mg/ml, he also showed that the extracts were slower and required longer time periods in terms of five to seven hours. The findings are also in agreement with Cabral et al., [66] who achieved 100% in-vitro action against Strongyloides stercoralis using 566mg/ml of Carica papaya L. extracts. Increasing the concentration several folds reduced the action time in the in-vitro assays.

4.4. In-vivo Efficacy tests of the extracts, Piperazine and Levamisole

All extracts were effective because they caused above 50% faecal egg count reductions; however the Carica papaya L. extracts were superior to those of Capsicum annuum L. CPLa were distinctly superior to CPLe in faecal egg reductions. The superior action was attributed to the vitamin C in presence of other anthelmintic compounds. The action of vitamin C on helminths in presence of various types of anthelmintics is not well known although Sengupta et al., [67] mentions that the observed actions arise from vitamin C enhancing the body defence lines.The findings about the in vivo efficacy of Carica papaya L. extracts are in agreement with Sen et al. [65]. The findings regarding the in-vivo action of Capsicum annuum L. extracts are in agreement with Gentiles et al. [64] who observed significant faecal egg reductions.

CPLa was as effective as levamisole, was different from CPLe, CPLa had higher concentration of vitamin C. There is need to investigate how vitamin C changes the action of synthetic and herbal anthelmintics. All extracts had higher FECR than piperazine citrate and levamisole hydrochloride was the highest faecal egg count reduction. The results show that there is no levamisole hydrochloride anthelmintic resistance in chicken in the study area although piperazine citrate anthelmintic resistance is likely. Chicken owners are advised to adopt the ethnoveterinary approaches in lieu of piperazine-citrate if levamisole hydrochloride is not accessible.

4.5 Future work

Pyranones were in relatively high concentrations in both CAF and CPL, it’s imperative to evaluate them for possible anthelmintic action. There is urgent need for comprehensive extract toxicity studies before they are purified and recommended for industrial scaling up. The role of vitamin C in anthelmintic actions requires further investigation to explore all opportunities in other herbal and synthetic combinations.

CD: Cell differentiation

DRC: Democratic republic of Congo

EVP: Ethnoveterinary Practice

FAO: Food and Agricultural Organization

FC: Frequency of citation

FCR: Food conversion ratio

FDA: Food and Agriculture Organization

FGD: Focus group discussion

GC-MS: Gas Chromatography-Mass Spectrometry

ICF: Informant consensus factor

MAAIF: Ministry of Agriculture, Animal industry and fisheries

NDA: National drug authority

OIE: Office international des epizooties

PRK: Percentage of respondents with knowledge

QDA: Qualitative data analysis

SPP: species

UK: United Kingdom

Availability of data and materials

The datasets supporting the conclusion of this article are included with in the article and its additional files.

Consent of Publication

Not applicable.

Competing interests

The author declares no competing interests whatsoever.

Clinical Trial Number

Not Applicable

Funding

Acknowledgement

Author’s contributions:

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No competing interests reported.

GCMSdetails.pdf

Extracts of carica papaya L. and capsicum annuum L. showed comparable efficacy to piperazine citrate and levamisole hydrochloride in treatment of poultry helminths

Status:

Journal Publication

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials and Methods

2.1 Description of study area

2.2 Study design

2.3 Description and care of experimental chicken

2.4 Preparation of acetone and ethanolic extracts

2.5 Phytochemical and GC_MS analysis of plant extracts

2.6 In-vitro susceptibility tests

2.7 In-vivo efficacy experiment

2.8 Statistical Analysis

2.9 Ethical considerations

Results

3.1. Qualitative Phytochemical analysis

3.2. GC-MS profile of Carica papaya L. and Capsicum annuum L. extract

3.3. In-vitro Efficacy

3.4. In-vivo Efficacy

Discussions

4.1 Qualitative Phytochemical analysis of Carica papaya L. and Capsicum annuum L.

4.2 Quantitative Phytochemical analysis of Carica papaya L. and Capsicum annuum L.

4.3. In-vitro Efficacy tests of the extracts

4.4. In-vivo Efficacy tests of the extracts, Piperazine and Levamisole

4.5 Future work

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1