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In vitro anthelmintic activity of clove-leaf extract (Syzygium aromaticum) against Ascaridia galli

Santika Anggrahini, Irkham Widiyono1, Soedarmanto Indarjulianto1 and Joko Prastowo2

Faculty of Veterinary Medicine, Universitas Gadjah Mada, 55281 Bulaksumur Sleman, Yogyakarta, 55281, Indonesia
1 Department of Veterinary Internal Medicine, Faculty of Veterinary Medicine, Universitas Gadjah Mada, 55281
Bulaksumur Sleman, Yogyakarta, 55281, Indonesia
2 Department of Parasitology, Faculty of Veterinary Medicine, Universitas Gadjah Mada, 55281
Bulaksumur Sleman, Yogyakarta, 55281, Indonesia


Parasitic worms that live and develop in the gastrointestinal tract are often found in poultry. Ascaridia galli is the most common type of these worms. Commercial anthelmintic drugs cause many problems including environmental pollution, adverse effects on host health, and widespread anthelmintic resistance. As an alternative, clove leaves are very valuable as an anthelmintic because they contain eugenols, tannins, saponins, flavonoids. This study aims to determine the anthelmintic potential of the clove-leaf ethanol extract (CLE) against Ascaridia galli. A total of 240 worms were divided into 8 groups to be treated with CLE at seven different concentrations (20, 40, 60, 80, 100, 120, and 140 mg/mL) and saline as control, respectively with 3 repetitions. Ten worms of each group submerged into 25ml of the test solution for 3-9 hours, then evaluated using a Scanning Electron Microscope (SEM). The results showed the highest mean mortality at 3h, 6h, and 9h observation was CLE 140 mg/mL. The ethanol extract of clove leaves caused surface changes resulting in damages to the cuticle of A. galli adult worm. It can be concluded that CLE possesses anthelmintic activity.

Keywords: herbal, SEM, worm ultrastructure


Parasitic worms that live and develop in the gastrointestinal tract are often found in poultry. Ascaridia galli is the most common type of these worms (Fahrimal and Raflesia 2002). The prevalence of chickens infected with Ascaridia galli can reach 92% (Offionga et al 2013). Ascaridia galli can be found in broilers, layers, local chickens, and ducks (Jayentakumar Singh and Mohilal 2017). Many commercial anthelmintic drugs are used to control the incidence of ascaridiosis (Mubarokah et al 2019).

Commercial anthelmintic drugs cause many problems including environmental pollution, adverse effects on host health, and widespread anthelmintic resistance (Vercruysse et al 2011). The exploitation of medicinal plants provides a culturally acceptable alternative disease control option as well as ecological and environmentally friendly (Nalule et al 2011). Plants of the genus Syzygium from comparative studies revealed that they can have gastrointestinal activity against infectious agents, as anthelmintics (Patel et al 2010).

Clove (Syzygium aromaticum) is known as a native Indonesian plant that is used in kretek cigarette, food, beverage, and medicinal industries (Cortés-Rojas et al 2014; Jannah et al 2020). Nowadays it is also cultivated in India, Madagascar, Sri Lanka, the South of China, Tanzania, and other countries in the world (Bhuiyan et al 2010; Kamatou et al 2012). Generally, cloves are marketed in 2 main products: dried clove shoots and essential oil extracted from shoots, leaves or stems (Vernin et al 1994). In traditional Chinese medicine, clove oil has been used as a carminative, antispasmodic, antibacterial, and anthelmintic, while the buds are used to treat dyspepsia, gastritis, and diarrhea (Barceloux, 2008; Harborne and Baxter 1993). The most important active compound in clove essential oil is eugenol (Srivastava et al 2017).

Clove leaves are very valuable as an anthelmintic because they contain eugenols, tannins, saponins, flavonoids. Clove leaves contain about 1–4% essential oil. The composition of the resulting essential oil varies depending on the state of the clove leaves and the way of distillation, the resulting oil usually contains eugenol between 80 - 88%. Clove-leaf oil in Indonesia has a concentration of 75-77% eugenol (Razafimamonjison et al 2014). The phytochemical content of clove-leaf powder contains saponins, tannins, alkaloids, glycosides, and flavonoids (Nurdjannah 2004; Nurdjannah et al 1993).

Therefore, this study was conducted to determine the anthelmintic potential of clove-leaf ethanol extract (CLE) against Ascaridia galli through the proportion of phytochemical content of clove-leaf ethanol extract (CLE) and the effect of CLE the mortality rate and ultrastructure of adult Ascaridia galli.

Materials and methods

This research was conducted in November 2019 and observed in Laboratory of Veterinary Internal Medicine, Faculty of Veterinary Medicine, Universitas Gadjah Mada. Clove leaves were obtained from Magelang Regency, Central Java Province.

Ethical approval

Ethical approval was not applicable in this study as samples used for to collect Ascaridia galli were sourced from slaughtered chickens from poultry abattoirs.

Phytochemical tests
Tannin test

Samples were weighed at ± 50 mg, extracted with 10 mL diethyl ether for 20 hours, and filtered. The remaining diethyl ether was then evaporated. Distilled water was added to the sample to reach the volume of 10 mL. The tests used 1 mL of sample, in which 0.1 mL of Folin-Ciocalteu reagent was added and vortexed for 5 minutes. The solution was added with 2 mL of 20% sodium carbonate, vortexed for 5 minutes, added with distilled water to a volume of 10 ml, diluted 5 times and read the absorbance at λ 760 nm after incubation for 30 minutes at room temperature (Chanwitheesuk et al 2004).

Saponin test

The sample was weighed at 50 mg, added with 1 ml of water, and extracted in a vortex for 5 minutes. The solution was added with 50 µl of anisaldehyde, shaken, incubated for 10 minutes at room temperature, then added with 2 ml of 50% sulfuric acid, heated in a water bath at 60°C for 10 minutes. The solution was then added with water to a volume of 10 ml, diluted 10 times, then the absorption was read at λ 435 nm.

Flavonoid test

The sample was weighed at 0.10 g and put into a 10 ml test tube containing 0.3 ml of 5% sodium nitrite. After 5 minutes, the sample was added with 0.6 ml of 10% aluminum chloride. After another 5-minute incubation, 2 ml of 1 M sodium hydroxide and distilled water were added to bring the volume to 10 ml. The solution was diluted 25x, then the absorption was read at λ 510 nm.

Phenol test

Samples were diluted with 0.5 ml of Folin-Ciocalteu reagent and 7.5 ml of bi-distilled water. The mixture was incubated for 10 minutes at room temperature to allow complete mixing, then added with 1.5 ml of 20% sodium carbonate and bi-distilled water to a volume of 10 ml. The solution was diluted as needed, then the absorption was read at λ 760 nm.

In vitro test
Extract preparation

The clove leaves obtained were dried for 30 to 36 hours and then oven-dried for an additional 6 hours at a temperature of 45˚C. Samples that had been dried were ground to produce flour/powder measuring at 60 mesh. A total of ± 1 kg powder would be extracted by maceration/immersion technique. The solvent used was 96% ethanol. The ratio of the sample in the form of flour to ethanol was 1: 3. Soaking was done in the shaker for 2 hours, then incubated for an additional 12 hours. The extraction results were filtered, placed in a rotary evaporator to remove the ethanol, and oven-dried at 60° C for approximately 24 hours to obtain a dry extract.

In vitro tests

To study the effect of clove-leaf extract (CLE) on Ascaridia galli , extracts (initially dissolved in DMSO) were diluted in distilled water at the desired concentrations for use in different assays. A total of 240 worms were divided into 8 groups, CLE was prepared at seven different concentrations (20, 40, 60, 80, 100, 120, and 140 mg/ml) and saline as control, respectively with 3 repetitions. Ten worms in each group were submerged into 25ml of the tested solution for 3-9 hours, then evaluated using a Scanning Electron Microscope (SEM).

Scanning Electron Microscopy (SEM)

The specimen preparation process consisted of 5 stages, starting from cleaning the worms by soaking in a cacodylate buffer for 2 hours followed by agitation in an Ultrasonic cleaner for 5 minutes. Afterwards, these samples went through prefixation, fixation, dehydration, and drying. The dried samples were placed in a test tube, coated with copper in an ion coating machine for 15 minutes and observed with a Scanning Electron Microscope.

Statistical analyses

Mortality data obtained were statistically analyzed using PROC FREQ in SAS version 9.0. and continued with Tukey test for any differences detected.

Results and discussion

Table 1 shows the percentage of phytochemical contents of clove leaves extracted using ethanol.

Table 1. Phytochemicals of clove-leaf extract


% w/w









Based on table 1, it shows the percentage concentration of phytochemicals of clove leaves ethanol extract with a tannin content of 2.39%, 6.97% saponins, 6.39% flavonoids, and a total phenol of 18.0%.

Table 2. Mean percent mortality of Ascaridia galli after immersion for 3-9h


Mortality (%)


p value




Saline water


































a,b,c,d Different superscripts in column and row indicate significant differences (p<0.05)

The treatment of various concentrations of CLE solution showed significant differences at concentrations of 80, 100, 120, 140 mg/mL (Table 2). Meanwhile, the time of death showed a significant difference at 3h, 6h, and 9h. CLE at the concentration of 20 mg/mL did not show a significant difference from the control. The highest percentage of worm mortality was in treatment with the concentration of 140 mg/mL after 9h immersion.

The active phytochemical components found in clove leaves comprise of alkaloids, tannins, saponins, flavonoids, phenols, glycosides, and terpenoids (Madubuike et al 2018). The amounts of these compounds are influenced by various factors, namely species, varieties, growing conditions, seasonal variations, processing, and storage methods (Pyo et al 2014). The present study carried out on the ethanol extract of clove leaves (Syzygium aromaticum) revealed that there is a presence of medicinal active constituents: 2.39% tannins, 6.97% saponins, 6.39% flavonoids, and 18.04% phenol. The phenol quantification is an important step in drug discovery (Madhavan and Tharakan 2020).

Meanwhile, the beneficial effects of medicinal plants usually result from secondary products present in plants, usually not attributed to only one compound but a combination of metabolites (Parekh et al 2005). However, clove oil is known to contain several compounds other than eugenol (up to about 85-90%) that have antifungal, anesthetic and antiseptic properties, such as eugenol acetate that makes up 9-10% (Madubuike et al 2018). Several studies have revealed that eugenol is an antiparasitic agent with potential effects on the growth, viability, and morphology of different parasites, such as Giardia lamblia, Leishmania donovani, and Trypanosoma cruzi (Machado et al 2011; Islamuddin et al 2014; De Morais et al 2014; Santoro et al 2007)

Figure 1. The ultrastructure of Ascaridia galli not soaked in clove-leaf extract (CLE) (a1: cuticle in posterior
region of body, b1, c1: anterior end), destruction of the ultrastructure of Ascaridia galli soaked in CLE
at a concentration of 140 mg/mL (a2: cuticle in posterior region of body, b2, c2: anterior end)

As A. galli mortality rate is demonstrated in Table 2, Figure 1 shows the damage and wrinkling to the anterior side of the worm observed in SEM. This proves that there is anthelmintic activity of CLE ( Syzygium aromaticum) against Ascaridia galli. The anthelmintic activity of these plant extract is related to the presence of biologically active metabolites such as condensed tannins, flavonoids, steroids, terpenoids, alkaloids and saponins (Hoste et al 2006; Vargas-Magaña et al 2014). Tannins can attack A. galli either directly or indirectly. Tannin affects A. galli directly by attaching to the cuticle (Spiegler et al 2017). Tannin and two flavonoid compounds, namely quercetin and luteolin, work together to inhibit the growth of L3 H. concortus larvae in vitro (Zhong et al 2014). The antioxidants present in phenol have anthelmintic efficacy in stimulating worm death (Klongsiriwet et al 2015). For nematodes, saponins have been associated with the formation of complexes with cellular membrane components present at various stages of the nematode life cycle, leading to increased membrane permeability and causing these parasites to die (Srivastava et al 2017; Doligalska et al 2011). It was reported that the steroid saponin-rich and triterpenic fraction of Agave sisalana had an 'in vitro' ovicidal effect against goat nematodes (Vo et al 2017).


Clove-leaf ethanol extract (CLE) contains active compounds that can cause death and ultrastructural changes in Ascaridia galli. Therefore, clove leaves have the potential to be anthelmintic.


This study was supported by The Ministry of Education, Culture, Research, and Technology of the Republic of Indonesian through Pendidikan Magister Menuju Doktor Untuk Sarjana Unggul (PMDSU). The authors also thank to Universitas Gadjah Mada, Indonesia for the support.


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