Citation of this paper

Potential of Carica pubescens fruit peel as an alternative method to control Haemonchus contortus in small ruminants

Z A Baihaqi¹, I Widiyono² and W Nurcahyo³

¹ Student of Doctoral program of Veterinary Science, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia
zein.ahmad.b@mail.ugm.ac.id
² Department of Internal Medicine, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia
³ Department of Parasitology, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia

Abstract

Haemonchus contortus is the most dangerous nematodes on small ruminant in Indonesia, in other sectors, Carica agroindustry in Indonesia produces a lot of fruit husk waste which causes environmental pollution. This study aimed to evaluate the potential of Carica pubescens peel waste as an alternative method of control the nematode Haemonchus contortus. The total phenol was measured by the Folin-Ciocalteu method and flavonoid contents were identified using the colourimetric method. The parasites were observed with an S450 scanning electron microscope (SEM). Statistical analysis was performed using SPSS 21.0 software, two-way ANOVA followed by Tukey test to detect significant differences (p<0.05). The result was expressed as the mean ± standard deviation (SD). The total phenolic and flavonoid contents in the aqueous extract of fruit peel (AEFP) were higher than those in the ethanol extract of fruit peel (EEFP). An in vitro study showed that AEFP had a significantly (p= 0.00) greater ability to kill 100% of H. contortus at a lower concentration 2.5% after 5 h, whereas EEFP had 100% mortality at a concentration of 5% after 12 h. Ivermectin caused 100% mortality after 1 h. The results of SEM imaging showed damage to the cuticle of H. contortus. In conclusion, the fruit peel wastes of Carica pubescens have anthelmintic activity against H. contortus.

Keywords: anthelmintic, motility inhibition, SEM images

Introduction

Haemonchus contortus is one of the most pathogenic gastrointestinal nematodes (GINs) in small ruminants (Zhang et al 2019). H. contortus is a parasitic worm of the abomasum, particularly in sheep and goats. These worms have economically important effects on livestock in both tropical and sub-tropical regions worldwide (El-Ashram et al 2017). Heavy GINs infestation is associated with reduced growth rate, high treatment cost, decreased sales prices, and loss due to mortality (Araujo et al 2019). H. contortus is known to occur in Indonesia and high prevalence has been recorded in Wonosobo Regency during the dry and rainy seasons (Baihaqi et al 2019). The tropical environmental conditions in Indonesia constitute an ideal habitat for a variety of parasitic species. Animals are usually treated against using chemical products, which may lead to various resistance-related problems.

Recently, there have been problems related to chemical anthelmintic resistance, which has become a global issue (Costa et al 2008; Ferreira et al 2013). This has created a challenge for researchers to find active plant compounds to replace synthetic anthelmintics (Grando et al 2016). Barone et al (2018) stated that secondary plant metabolites can be used to control GINs in small ruminants. Sambodo et al (2018) reported that the interaction between the active compound Biophytum petersianum and Haemonchus contortus causes changes in the structure of the cuticle and will have an impact on the inhibition of the motility of the parasite and the disturbance of the nematode’s nutrition, which might eventually lead to worm undernourishment.

The final waste disposal of the fruit industry in Indonesia is mostly by landfill, which causes pollution. One type of fruit that is commonly found in several highlands in Indonesia and is processed for the Indonesian agro-industry is Carica pubescens. This fruit contains active compounds in the form of flavonoids, alkaloids, and phenolic compounds (Rahayu et al 2019). Fruit peels are a rich source of cellulose, hemicellulose, phenolic compounds, and terpenes, and thus have the potential to be used as an animal feed and herbal anthelmintic (Joglekar et al 2019). The anthelmintic potential of the compounds contained in Carica pubescens peel has not yet been evaluated. This study focused on the use of Carica pubescens fruit extract as an alternative to chemical anthelmintics against GINs in small ruminants.

Materials and methods

Ethical approval

This research was approved by the Institutional Ethical Committee, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia. Number: 0013/ EC-FKH/Int./2019.

Plant and extraction materials

The fruit peel of Carica pubescens was obtained from the Carica fruit industry in Wonosobo Regency, Central Java, Indonesia. The fruit peel is washed with water and being aerated, then being cut into smaller pieces and put in the oven at 55^oC for 5 days. The sample is ground into powder, soaked with aqueous or ethanol solvent and allowed to stand for 24 hours. The samples are filtered between the pulp and the solvent. The solvent is evaporated until it runs out and becomes a thick extract.

Identification of plant phytochemicals

The active compounds contained in C. pubescens, namely phenols, tannins, alkaloids, flavonoids, and steroids, were qualitatively identified using the method described by Kanagavalli et al (2018), while the total phenol was measured by the Folin-Ciocalteu method and the results were expressed as mg gallic acid equivalents (GAE) (Gunes et al 2019) and flavonoid contents were identified using the colourimetric method and the results were expressed as mg rutin (RE) (He et al 2019).

In vitro motility inhibition of adult worm

Adult female H. contortus worms were collected from a Godean sheep slaughterhouse in Yogyakarta. After slaughtering the sheep, the abomasum is brought to the parasitology laboratory and H. contortus is collected directly. In vitro, anthelmintic research was carried out in February 2020 on the laboratory of internal medicine, Animal Hospital Prof. Soeparwi, Faculty of Veterinary Medicine, Universitas Gadjah Mada. An in vitro adult worm mortality assay was performed by modifying the method of Sambodo et al (2018). The number of worms used in each concentration is twenty. Treatments were carried out using various concentrations of Carica pubescens fruit peel extract (0; 0,2; 0,4; 0,8; 1; 2,5 and 5 %), and 1% ivermectin as a positive control. The test was repeated 3 times. Worm mortality was confirmed by physical stimulation, i.e., touching the worm’s body using tweezers and keeping it in lukewarm water for 5 minutes before declaring it dead. Time of mortality of each worm was recorded.

Scanning electron microscopy

contortus obtained from the in vitro assays studies were fixed with 2% glutaraldehyde solution in a 0.1 M sodium cacodylate buffer for 4 h at 4°C. The worms were dehydrated with ethanol, then critical point dried with an EMSCOPE CPD 750 and coated with gold-palladium for 5 min. The parasites were then observed using an S450 scanning electron microscope (Hitachi, Japan) at an accelerating voltage of 15 kV.

Statistical analysis

The results of the in vitro Haemonchus contortus mortality assay were recorded and analyzed by SPSS 21.0. We performed a two-way ANOVA followed by the Tukey test to detect significant differences p<0.05). The results were expressed as the mean ± standard deviation (SD) (p<0.05).

Results

The qualitative phytochemical analysis of Carica pubescens fruit peel waste with 70% aqueous and ethanol solvents is presented in Table 1.

Extract	Tannin	Flavonoid	Alkaloid	Saponin	Steroid
Table 1. Qualitative phytochemical analysis of Carica pubescens fruit peel

Aqueous	+	+	-	+	-
Ethanol 70%	+	+	+	-	-

Fig 1. Total phenol (mg GAE/g dw) and flavonoid (mg RE/g dw) of Carica pubescens

AEFP, the aqueous extract of fruit peel; EEFP, ethanol extract of fruit peel;
GAE, gallic acid equivalent; RE, rutin equivalent; dw, dry weight

The AEFP of Carica pubescens contains tannins, flavonoids, and saponins, while the EEFP contains tannins, flavonoids, and alkaloid. Figure 1 shows the total phenol and flavonoid contents contained in the fruit peel of Carica pubescens with 70% aqueous and ethanol solvents. The total phenolic and flavonoid contents were 8.7 mg gallic acid equivalent (GAE)/g dw and 4.2 mg rutin equivalent (RE)/g dw in the AEFP of C. pubescens, while those in the EEFP were 6.3 mg GAE/g dw and 4.1 mg RE/g dw, respectively.

Treatment	Time of death (h) - A.E.F.P. C. pubescens								SEM	p value
Table 2. In Vitro Haemonchus contortus mortality test (A.E.F.P. C. pubescens)
Treatment	0.5	1	2	3	4	5	6	12	SEM	p value

0%	0.00±0.00^Aa	0.00±0.00^Aa	0.00±0.00^Aa	0.00±0.00^Aa	0.00±0.00^Aa	0.00±0.00^Aa	0.00±0.00^A	0.00±0.00^Aa	0.00	-
0,2%	0.00±0.00^Aa	0.00±0.00^Aa	0.00±0.00^Aa	0.00±0.00^Aa	8.35±1.16^Aa	23.35±0.58^Bb	31.65±1.16^Bbc	41.65±1.53^Bc	3.35	0.00
0,4%	3.35±1.16^Aa	8.35±0.58^ABa	8.35±0.58^ABa	16.65±0.58^Bab	26.65±0.58^Bbc	36.65±1.53^Ccd	48.35±1.16^Cde	61.65±1.53^Ce	4.21	0.00
0,8%	6.65±1.16^ABa	16.65±0.58^ABab	23.35±0.58^BCabc	26.65±0.58^Cbc	36.65±1.16^Cde	48.35±0.58^De	66.5±1.16^Df	81.65±2.89^CDf	5.03	0.00
1,0%	6.65±0.58^ABa	21.65±1.53^BCb	28.35±1.53^Cbc	36.65±0.58^Dc	58.35±0.58^Dd	61.65±0.58^Ede	73.35±0.58^Def	83.35±0.58^DEf	5.31	0.00
2,5%	16.65±0.58^Ba	23.35±1.16^BCab	31.65±1.53^Cb	68.35±1.16^Eb	93.35±0.58^Ed	100.00±0.00^Fd	100.00±0.00^Ed	100.00±0.00^Ed	7.23	0.00
5%	16.65±0.58^Ba	36.65±1.53^Ca	48.35±2.09^Da	93.35±0.58^Fa	100.00±0.00^Ea	100.00±0.00^Fa	100.00±0.00^Ea	100.00±0.00^Ea	6.81	0.00
Ivermectin 1 %	43.35±1.53^Ca	100.00±0.00^Db	100.00±0.00^Eb	100.00±0.00^Fb	100.00±0.00^Eb	100.00±0.00^Fb	100.00±0.00^Eb	100.00±0.00^Eb	3.92	0.00
SEM	2.88	6.38	6.48	7.79	8.04	7.54	7.17	6.96	-	-
p value	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	-	-

Treatment	Time of death (h) - E.E.F.P. C. pubescens								SEM	p value
Table 3. In vitro Haemonchus contortus mortality test (E.E.F.P. C. pubescens)
Treatment	0.5	1	2	3	4	5	6	12	SEM	p value

0%	0.00±0.00^Aa	0.00±0.00^Aa	0.00±0.00^Aa	0.00±0.00^Aa	0.00±0.00^Aa	0.00±0.00^Aa	0.00±0.00^Aa	0.00±0.00^Aa	0.00	-
0,2%	0.00±0.00^Aa	0.00±0.00^Aa	11.65±1.16^ABab	13.35±1.53^ABCabc	16.65±1.53^BCbc	23.35±0.58^BCbc	28.35±1.53^BCcd	41.65±1.53^Bd	2.93	0.00
0,4%	0.00±0.00^Aa	0.00±0.00^Aa	3.35±0.66^ABab	6.65±1.53^ABab	11.65±0.58^ABabc	13.35±0.58^ABbc	21.65±0.58^Bc	36.65±1.53^Bd	2.57	0.00
0,8%	0.00±0.00^Aa	1.65±0.57^Aab	6.65±1.16^ABabc	16.65±0.58^BCbcd	18.35±0.58^BCcd	26.65±1.16^BCd	31.65±2.08^BCde	46.65±1.53^Be	3.28	0.00
1,0%	1.65±0.58^Aa	3.35±1.16^Aa	11.65±1.16^ABab	21.65±0.58^Cbc	26.65±0.58^Dbc	31.65±1.53^CDcd	43.35±1.53^Cde	48.35±1.53^Be	3.53	0.00
2,5%	6.65±1.53^Aa	11.65±0.58^Aab	16.65±1.53^Babc	21.65±1.53^Cabcd	28.35±0.58^Dbcde	33.35±1.53^CDcde	36.65±0.58^BCde	43.35±1.53^e	2.72	0.00
5%	11.65±1.53^Aa	13.35±1.16^Aa	16.65±1.53^Ba	21.65±0.58^Ca	26.65±1.53^Dab	43.35±1.53^Db	78.35±1.16^Dc	100.00±0.00^Cd	6.53	0.00
Ivermectin 1 %	43.35±1.53^Ba	100.00±0.00^Bb	100.00±0.00^Cb	100.00±0.00^Db	100.00±0.00^Eb	100.00±0.00^Eb	100.00±0.00^Eb	100.00±0.00^Cb	3.92	0.00
SEM	3.00	6.71	6.42	6.15	5.97	5.87	6.33	6.58	-	-
p value	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	-	-

In vitro tests showed that the AEFP of Carica pubescens started to inhibit H. contortus at a concentration of 0.4% at 0.5 h (Table 2), while the EEFP was observed to start inhibiting at a concentration of 0.2% at 2 h into the experiment (Table 3). The AEFP of Carica pubescens significantly (p=0.00) suppressed 100% of worms at a concentration of 2.5% at 5 h (Table 2), while the EEFP of Carica pubescens at a concentration of 5% at 12 h (Table 3). The anthelmintic activity of the ivermectin treatment (positive control) started to take place at 1 h and successfully immobilized 100% of worms (p=0.00) at 1 h.

Scanning electron microscopy image Fig02

(A) Scanning electron microscopy (SEM) image of Haemonchus contortus (buccal cavity) without extract treatment. (B) SEM image of H. contortus (buccal cavity) were observed to be damaged at the buccal area after treatment with the aqueous extract of fruit peel (AEFP) of Carica pubescens. (C) SEM image of H. contortus (buccal cavity) were observed to be damaged at the buccal area after treatment with the ethanol extract of fruit peel (EEFP) of C. pubescens. (D) SEM image of H. contortus (body) without extract treatment. (E) SEM image of H. contortus (body) shows an aggregate buildup in the annular cuticles after treatment with the
AEFP of C. pubescens. (F) SEM image of H. contortus (body) shows wrinkles in the annular cuticles after treatment with the EEFP of C. pubescens

Fig 2. SEM images of the anterior end and the cuticle of the adult female H. contortus

Discussion

Plants are commonly used as an anthelmintic alternative because they contain secondary metabolites. The findings from the research of Sebai et al (2020) state that plant secondary metabolite compounds are responsible for worm damage until death. Hoste et al (2012) add that high immobility in adult worms is probably linked to the damage caused by protein–tannin complexes to the cuticle of the nematode. Fiel et al (2017) stated that the excessive use of synthetic drugs for anthelmintic is leading to increased occurrence of anthelmintic resistance. Cortes-Morales et al (2019) add that interest in the use of natural plant compounds towards anthelmintic activity, such as plant extracts and plant derivatives to find valid alternatives as substitutes for synthetic drugs at this time.

Rahayu et al (2019) added that the different types of active compounds when using different solvents are because of the characteristics of the active compounds, whether they are polar or nonpolar. Ugbogu et al (2019) stated that secondary metabolites are plant natural products have the potential to animal health alternative and improve productivity, improve rumen fermentation, reduce the loss of feed energy, increase animal lifetime performance, and reduce greenhouse gases production- CH₄ and CO₂during animal production.

Fruit peel waste of Carica papaya is a plant waste that potentially contains secondary metabolites. Rehab et al (2017) stated that plants are an important source for the discovery of new products of medicinal value for drug development and plants secondary metabolites are unique sources for pharmaceuticals. Root, stem, leaves, fruits, and flowers of various plants were found to possess secondary metabolites to show bioactivity. This is in line with a study by Rahayu et al (2019) who reported that phenolic compounds, alkaloids, and flavonoids were found in the leaves of Carica pubescens with methanol, ethyl acetate, and chloroform solvent extractions. Shakya et al (2016) added that the secondary metabolites of plants include saponins, flavonoids, alkaloids, terpenoids, steroids, glycosides, tannins, and essential oils. As such, Carica pubescens has potential as a herbal medicinal ingredient. Sen et al (2020) reported that plants commonly used as an alternative medicine generally contain polar substances, also known as plant bioactive components or secondary metabolites, including phenolic compounds and flavonoids. Several studies have confirmed that some secondary metabolites contain anthelmintic activity against the parasite H. contortus (Marie-Magdeleine et al 2008; Acharya et al 2014; Kommuru et al 2014; Ribeiro et al 2015; Ferreira et al 2018).

The AEFP of Carica pubescens was observed to start inhibition earlier and successfully kill 100% of H. contortus earlier than EEFP. This is because AEFP has a quantitatively (8.7 mg GAE/g dm) higher content of phenolic compounds than EEFP (6.3 mg GAE/g dm). Also, saponins in AEFP increase the anthelmintic potential of the preparation. The extract of Ipomoea imperati (Vahl) Griseb, which contains phenolics and flavonoids, successfully inhibited H. contortus (Araujo et al 2019). Adnan et al (2019) added that phenolic compounds play a role in anthelmintic potential.

Montellano et al (2013) stated that worms exposed to active compounds will experience damaged cuticles, buccal cavities, esophagi, and reproductive tracts. Barone et al (2018) stated that damage and aggregates were found in the buccal region of H. contortus which came into contact with condensed tannins from cranberry vines, while worms not exposed to active compounds were observed to be normal. Doligalska et al (2011) stated that the interaction between saponins from plants with the cell membranes of GINs could induce the formation of micelle-like aggregates that interfere with functions, causing lysis. Saponins will increase the membrane permeability, causing parasites to die.

Conclusion

The fruit peel waste of Carica pubescens is a potential alternative for suppressing the gastrointestinal nematode H. contortus.
AEFP and EAFP can suppress 100% H. contortus.
AEFP and EAFP are proven to damage the anterior end and the cuticle of the adult female H. contortus.

Acknowledgements

This study was supported by the Ministry of Research, Technology and Higher Education of the Republic of Indonesian through Pendidikan Magister Menuju Doktor Untuk Sarjana Unggul (PMDSU) Program Grant Number 148/SP2H/PTNBH/DPRM/2018.

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Received 8 May 2020; Accepted 14 May 2020; Published 1 July 2020

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