Research Article

In Vitro Anthelmintic Activity of Tectona grandis Linn. Leaves Nanoemulsion

Dian Indriati1, Yulita Nadzifa Erista1, Irkham Widiyono2*, Zein Ahmad Baihaqi3, Yanuartono2

1Faculty of Veterinary Medicine, Universitas Gadjah Mada, Bulaksumur Sleman, Yogyakarta, Indonesia; 2Department of Internal Medicine, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Bulaksumur Sleman, Yogyakarta, Indonesia; 3Research Center for Animal Husbandry, National Research and Innovation Agency (BRIN), Jakarta, Indonesia.

Received | January 29, 2025; Accepted | March 18, 2025; Published | May 15, 2025

*Correspondence | Irkham Widiyono, Department of Internal Medicine, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Bulaksumur Sleman, Yogyakarta, Indonesia; Email: [email protected]

Citation | Indriati D, Erista YN, Widiyono I, Baihaqi ZA, Yanuartono (2025). In vitro anthelmintic activity of Tectona grandis linn. Leaves nanoemulsion. J. Anim. Health Prod. 13(2): 346-354.

DOI | https://dx.doi.org/10.17582/journal.jahp/2025/13.2.346.354

ISSN (Online) | 2308-2801

Copyright © 2025 Kumar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Helminths are a diverse group of parasites that represent a significant health challenge for animals across the globe. Gastrointestinal worm infections are prevalent in ruminants and represent a significant barrier to their productivity (Hou et al., 2022). Among these infections, haemonchiasis stands out as a critical concern for livestock. The nematode Haemonchus sp. is recognized as the most pathogenic species affecting the gastrointestinal tract of sheep and goats globally. According to the Food and Agriculture Organization (FAO, 1991), the prevalence of haemonchiasis in goats in Indonesia is reported to be 89.4%, resulting in annual economic losses estimated at USD 1 million. The primary approach to managing helminth infections has been the use of pharmaceutical anthelmintics. However, the widespread and frequent use of these drugs has resulted in a marked increase in anthelmintic resistance. This resistance is characterized by the inherited reduction in sensitivity of a parasite population to an anthelmintic that was previously effective against it (Fissiha and Kinde, 2021).

The management and control of gastrointestinal nematodes in livestock production primarily rely on the application of macrocyclic lactone anthelmintics (Yanuartono et al., 2020). Consequently, this practice fosters selective pressure within practice populations, leading to the development of resistance against macrocyclic lactones (Gasbarre et al., 2009). Notably, widespread reports have documented resistance among ruminant livestock, particularly towards ivermectin (Macedo et al., 2015). Recent research has identified numerous phytochemical properties of various plants that can be utilized to address health issues in livestock. Plant-derived natural anthelmintics have gained popularity regarding the management of nematode infections in ruminant animals and are anticipated to mitigate the development of resistance to conventional anthelmintics in the future (Miro et al., 2020). Multiple studies have highlighted the potential of active compounds obtained from plant-based agro-industrial waste for alternative treatments in livestock. These compounds have been reported to act as bio-anthelmintics, bio-antibacterials, methane reduction agents, and productivity enhancers (Baihaqi et al., 2020; Sakti et al., 2024; Rokana et al., 2024; Prasetyo et al., 2024; Lokapirnasari et al., 2024; Prayudi et al., 2023; Lisnanti et al., 2023). Additionally, Baihaqi et al. (2023, 2024) also supported these findings.

Tectona grandis (T. grandis) is recognized for its medicinal significance, with numerous claims regarding its efficacy in treating severe illnesses according to traditional Indian medicine. The survey indicated that the plant is utilized for the treatment of urinary discharge, bronchial disorders, cold, and headaches. Additionally, it is employed as a laxative, sedative, diuretic, and anti-diabetic agent. The flowers of T. grandis are utilized in the management of bronchitis, biliousness, and urinary discharge. Both the flowers and seeds serve as diuretics, while the wood exhibits expectorant, anti-inflammatory, anti-bilious, and anthelmintic properties. The bark functions as a potent astringent, particularly in cases of bronchitis. The roots are employed for managing anuria and urinary retention. Additionally, nut oil is used in the treatment of scabies and other skin conditions, as well as for promoting hair growth (Asdaq et al., 2022). Ogunmefun et al. (2017) stated that T. grandis leaves contain secondary metabolites, namely alkaloids, flavonoids, tannins, saponins, cardiac glycosides, total phenolics, and carotenoids as listed in Table 1. According of Gururaja et al. (2011) the anthelmintic activity of the ethanolic extract derived from the fruits of T. grandis preliminary phytochemical analysis of the crude ethanolic extract indicated the presence of flavonoids. At a concentration of 50 mg/ml, the ethanolic extract induced paralysis in Pheretima posthuma within 21 minutes and resulted in mortality within 32 minutes (Gururaja et al., 2011).

 

Table 1: Secondary metabolite of Tectona grandis Leaves.

Phytochemical name	Presence in T. grandis leaf
Alkaloid (mg per 100 g)	1778.30
Flavonoids (mg per 100 g)	666.60
Tannins (mg per 100 g)	1583.00
Saponins (mg per 100 g)	406.60
Cardiac glycosides (mg per 100 g)	20.00
Total phenolics (GAE per g)	69.50
Carotenoids (µg per 100 g)	1268.33

 

adopted from Ogunmefun et al (2017).

 

Nanoemulsion is a thermodynamically stable lipid-based drug delivery system composed of oil, surfactants, cosurfactants, and water, characterized by droplet sizes in the nanometer range (Ahmad et al., 2019). Nanoemulsion have been reported to possess several advantageous properties, including high drug solubility, substantial protective capabilities, thermodynamic stability, enhanced oral bioavailability, and a droplet size smaller than 100 nm (Vatsraj et al., 2014). Currently, anthelmintics formulated as nanoemulsion are not widely available. Therefore, this study aims to determine the potential of T. grandis leaf nanoemulsion as an alternative anthelmintic for Haemonchus sp. worms in small ruminants.

MATERIALS AND METHODS

Ethical Clearance

The Institutional Ethics Committee of the Faculty of Veterinary Medicine at Universitas Gadjah Mada in Yogyakarta, Indonesia, has granted approval for the study under the reference number: 70/EC-FKH/int./2024

Area of Study and Collection of Samples

The study was carried out from July to November, 2024. The in vitro anthelmintic experiments were conducted at the Animal Parasitology Laboratory in the Department of Internal Medicine, Faculty of Veterinary Medicine, Universitas Gadjah Mada. Scanning electron microscopy were carried out at the Research Center for Animal Husbandry, National Research and Innovation Agency (BRIN), Indonesia. T. grandis leaves samples were collected from community forests located in Gunungkidul, Yogyakarta. Concurrently, adult Haemonchus sp. nematodes were collected from the abomasum of sheep that exhibited clinical symptoms, including thin, dull hair, and pale. The sheep selected for this study were sourced from the Mutiara Domba Slaughterhouse in Godean, Yogyakarta.The procedure involved carefully opening the abomasum with scissors and extracting the worm using tweezers, which were then placed in a sodium chloride (NaCl) solution for preservation. The collected specimens were specifically identified as female worms, characterized by alternating red and white coloration on their bodies (Victor et al., 2014). A total of 100 female worms were required for this experiment, sourced from seven different abomasums.

Extraction and Nanoemulsion

Collected and selected T. grandis leaves were first cut into smaller pieces and then dried in an oven at 55°C until fully desiccated. The dried leaves were subsequently ground into a fine powder. Ethanol (70%) was gradually added during the evaporation process using a rotary evaporator until the desired extract was obtained (Charoensit et al., 2021). The extract was then formulated into an oil-in-water nanoemulsion, where the oil phase is dispersed within the aqueous phase. The nanoemulsion was produced using a high-energy method with an ultrasonic homogenizer, resulting in smaller and more stable particle sizes (Sadeq, 2020). The T. grandis leaf nanoemulsion was prepared at three different concentrations: 3%, 5%, and 7%, with formulation details provided in Table 2. This formulation was adapted from the study by Sreeja and Poulose (2017). The formulation consists of leaf extract, virgin coconut oil (VCO) as the oil phase, Tween 80 as the surfactant, polyethylene glycol 400 (PEG 400) as a cosurfactant, chitosan as an antiparasitic agent, and distilled water as the aqueous phase.

 

Table 2: Formulation of Tectona grandis Leaves nanoemulsion.

Formulation of nanoemultion (100 ml)	Material*
	T. grandis Extract (gram)	VCO (ml)	Tween 80(ml)	PEG 400 (ml)	Chitosan (ml)	Distilled water (ml)
Concentration 3%	3	1	3	1	1	91
Concentration 5%	5	1	3	1	1	89
Concentration 7%	7	1	3	1	1	87

 

* VCO: virgin coconut oil; PEG 400: polyethylene glycol 400.

 

In vitro Test for the Mortality of Adult Worm

Adult female Haemonchus sp. were obtained from an abattoir in Yogyakarta. In vitro anthelmintic experiments were conducted using a combination of methodologies (Mondal et al., 2015). Haemonchus sp. worms were divided into three treatment groups: nanoemulsion at concentrations of 3%, 5%, and 7%; a negative control group with 0.9% sodium chloride; and a positive control group with 2 mg/ml albendazole, with 20 worms per group. Each group consisted of four repetitions. The worms were placed in 50 mm Petri dishes containing nanoemulsion, sodium chloride, or albendazole solution and incubated at 37°C for 0.5, 1, 2, 3, 4, 6, 8, and 12 hours. The Petri dishes were covered with a black cloth. The incubation period was recorded, and the number of surviving worms was noted. Mortality was confirmed by applying pressure to the body of the worm using tweezers. Worms that exhibited no movement were classified as deceased. A worm was considered dead when it lost its motility and showed a noticeable fading of color.

Scanning Electron Microscopy Analysis

Specimens of Haemonchus sp. collected from the in vitro assay studies were preserved in a 2% glutaraldehyde solution within a 0.1 M sodium cacodylate buffer for 4 hours at 4°C (Baihaqi et al., 2023). The worms were subsequently dehydrated using a gradient of ethanol from 30% to absolute concentration, critically dried with an EMSCOPE CPD 750 (Ashford, Great Britain), and coated with gold-palladium for 5 minutes. The parasites were then examined using a Hitachi S450 scanning electron microscope.

Statistical Analysis

The worm mortality data were evaluated using a one-way analysis of variance (ANOVA). Statistical variations among the means were evaluated using Duncan’s multiple-range test. Result were deemed statistically significant at p<0.05. The analyses were conducted using IBM Statistical Package for the Social Sciences (SPSS) version 30.0.

RESULTS

In vitro Test for the Mortality of Adult Worm

Table 3 presents the results of the mortality test on Haemonchus sp. The nanoemulsion of T. grandis leaves was found to be effective in causing the death of Haemonchus sp.

 

Table 3: In vitro Haemonchus sp. mortality test with Tectona grandis leaves nanoemulsion.

Treatment	Mortality of Haemonchus sp.								P value
	0.5 h	1 h	2 h	3 h	4 h	6 h	8 h	12 h
3%	0±0.00	0±0.00	0±0.00a	0±0.00a	15±1.0a	30±2.58b	60±1.63b	75±1.91b	0.001
5%	0±0.00	0±0.00	5±1.0a	10±1.15a	20±2.82a	55±1.0c	65±1.0b	90±1.15b	0.001
7%	0±0.00	0±0.00	20±0.00b	45±1.0b	100±0.00b	100±0.00d	100±0.00c	100±0.00b	0.001
NaCl	0±0.00	0±0.00	0±0.00a	0±0.00a	0±0.00a	0±0.00a	10±2.0a	15±3.0a	0.001
Albendazole	0±0.00	0±0.00	50±1.15c	100±0.00c	100±0.00b	100±0.00d	100±0.00c	100±0.00b	0.001
P value	-	-	0.001	0.001	0.001	0.001	0.001	0.001	-

 

 

Significant differences (P = 0.001) were recorded between various treatment groups for the mortality of Haemonchus sp. which was also affected by the incubation time (P = 0.001). At a concentration of 3%, the nanoemulsion starts to kill the worms after 4 hours. At concentrations of 5% and 7%, the nanoemulsion begins to cause worm mortality after 2 hours. The positive control, albendazole, kills all the worms within 3 hours. Compared to the positive control, the 7% T. grandis leaf nanoemulsion shows a similar effect, with all worms dying by the 4th hour.

Scanning Electron Microscopy

Figure 1 and 2 display scanning electron microscopy images of adult female Haemonchus sp. of the front region and cuticle. Haemonchus sp. has damage to the buccal area and cuticle. Figure 1 illustrates that worms immersed in sodium chloride (NaCl) solution, serving as the negative control, exhibit a smooth cuticle surface without any signs of damage. In contrast, those treated with albendazole (positive control) display damage in the buccal region, accompanied by the presence of aggregates. Furthermore, Haemonchus sp. worms subjected to T. grandis leaves nanoemulsion treatment at concentrations of (b) 3%, (c) 5%, and (d) 7%, demonstrated damage to the buccal area. Figure 2 shows worms immersed in sodium chloride (NaCl) solution, serving as the negative control, which exhibit smooth cuticles devoid of aggregates and display an undamaged reticular pattern on their longitudinal dorsal surfaces. In contrast, following treatment with teak leaf nanoemulsion at concentrations of 3%, 5%, and 7%, Haemonchus sp. worms exhibited wrinkling in the annular cuticle. Additionally, worms treated with albendazole (positive control) displayed numerous wrinkles in both the annular and dorsal longitudinal regions.

DISCUSSION

Secondary metabolites derived from plants are crucial in the realm of drug discovery and development. These compounds are extracted from plant materials through various extraction processes, which involve the separation of desired substances from their mixtures using specific solvents

 

(Mukhriani, 2014). In the case of teak leaves, a 70% ethanol solution is employed for extraction. The choice of solvent in the extraction process is primarily determined by its polarity, which influences the solubility of the target compounds (Hikmawanti et al., 2020). Ethanol is a polar solvent that is able to extract flavonoid compounds, which are also polar so they are easily soluble (Markham, 1988). Moreover, ethanol is favored for its safety profile, making it suitable for the extraction of phytochemicals and bioactive compounds from plants. It is less susceptible to microbial contamination by fungi and bacteria, does not introduce toxins, remains inert with respect to the extracted substances, and exhibits favorable absorption characteristics (Sa’adah and Nurhasnawati, 2017).

The ethanol extract of teak leaves is formulated into an oil-in-water (o/w) nanoemulsion, which consists of an oil phase dispersed within a water phase. The production of this nanoemulsion employs a high-energy technique utilizing an ultrasonic homogenizer, which is known to yield nanoemulsions with smaller and more stable particle sizes (Sadeq, 2020). The oil phase in this formulation is VCO, which serves as a solvent for lipophilic active substances (Puspitasari, 2022). In the emulsification process, the oil phase forms droplets within the dispersion medium, facilitated by surfactants and cosurfactants. Tween 80, a non-ionic surfactant characterized by its sorbitan ester bond resulting from the reaction between sorbitol and fatty acids with ethylene oxide, is utilized in this formulation (Amin and Das, 2019). The cosurfactant employed is PEG 400, a colorless and viscous liquid that is soluble in water. The role of the cosurfactant is to enhance the emulsification process and improve the stability of the nanoemulsion (Sadeq, 2020). Additionally, chitosan is incorporated into the formulation to inhibit the growth of bacteria and fungi in the nanoemulsion preparations (Chaudhary et al., 2020). Preliminary phytochemical analysis T. grandis Linn has revealed the presence of glycosides, alkaloids, carbohydrates, proteins, tannins, and flavonoids in the ethanolic extracts. T. grandis leaves methanol extract contains Sinapic, gallic, p-hydroxybenzoic, ferulic, p-coumarate, chlorogenic, cinnamic, vanillic acid (Murukan and Kumara, 2018). T. grandis leaves aqueous extract also contains Sinapic, gallic, p-hydroxybenzoic, ferulic, p-coumarate, chlorogenic, cinnamic, vanillic acid (Koffi et al., 2015).

T. grandis is recognized for its medicinal significance, with numerous reports indicating its efficacy in treating various diseases according to traditional Indian systems of medicine. Surveys indicate that this plant is utilized for the management of conditions such as urinary discharge, bronchitis, colds, and headaches. Additionally, it is employed in the treatment of scabies and is known for its laxative and sedative properties, as well as its diuretic, anti-diabetic, analgesic, and anti-inflammatory effects (Asdaq et al., 2022). The leaves of T. grandis are rich in secondary metabolites, which include alkaloids, tannins, saponins, flavonoids, anthraquinones, and carotenoids (Ogunmefun et al., 2017). Many studies have been undertaken to assess the efficacy of tannins as anthelmintics, both in vitro and in vivo. T. grandis leaves exhibits significant anthelmintic activity; however, the time required for inducing mortality in the worms is relatively prolonged. According to Javalgikar at al. (2019) T. grandis Linn caused paralysis within 19 to 32 minutes and resulted in death within 30 to 49 minutes. In comparison, the standard drug induced paralysis within 20 minutes and led to mortality at 48 minutes. These findings suggest that the test drug demonstrates a potent effect relative to the standard treatment. Toklo et al. (2021) reported that the evaluation of anthelmintic activity demonstrated that the extracts at various doses, along with all tested compounds at a concentration of 150 mg/ml, significantly inhibited larval migration and the motility of adult parasitic worms when compared to the negative control, which consisted of a phosphate buffer solution. The study conducted by Handayanta et al. (2023) demonstrated that a 12% of Acacia auriculiformis leaves achieved 100% mortality of Haemonchus contortus within six hours. This finding suggests that T. grandis leaves nanoemulsion exhibits a higher efficacy in eliminating Haemonchus sp. Another study consucted by Mumed et al. (2022) demonstrated that a 500 mg/ml extract of Croton macrostachyus leaves achieved 100% mortality of Haemonchus contortus within four hours. In the present study, 7% T. grandis leaves nanoemulsion exhibited a similar ability to eliminate Haemonchus sp. within the same timeframe. Generally, the capacity of tannins to bind proteins enables them to penetrate various structures of nematodes. The cuticle, in particular, is a structure that is easily penetrated by macromolecules like tannins. A series of microscopic analyses have demonstrated detrimental effects on the cuticle that also impact the underlying tissues across various nematode species (Greiffer et al., 2022).

In nematodes, saponins have been linked to the formation of complexes with components of cellular membranes at various stages of the nematode life cycle. This interaction results in increased membrane permeability, ultimately leading to the mortality of these parasites (Maestrini et al., 2019). Exposure to medicinal ethnoveterinary plant extracts induces characteristic alterations in the surface morphology of worms. This hypothesis is supported by numerous prior reports documenting the anthelmintic activity of plant extracts against helminths. The process induces membrane destabilization, alters membrane permeability, and results in the loss of membrane potential, ultimately leading to cellular lysis and damage to the cuticle. Flavonoids exhibited the most pronounced activity, with flavones showing particularly strong effects, suggesting their potential for further development into derivatives that may enhance anthelmintic efficacy (Balqis et al., 2017). Alkaloids possess neurotoxic properties by inhibiting acetylcholinesterase, leading to acetylcholine accumulation, which induces spasms in the body wall muscles, followed by flaccid paralysis and eventual death of gastrointestinal parasites (Hrckova and Velebny, 2013).

Nematodes are the sole group of parasites that have a robust protective outer layer called the cuticle. This cuticle is made up of a syncytium of proteins, primarily composed of collagen. The mortality of these worms is likely attributed to damage sustained in various body parts, including the buccal area and cuticle, as a result of contact with metabolites present in the nanoemulsion derived from T. grandis leaves. This structure functions not only as a protective barrier but also as the structural framework for the organism’s body (Lalthanpuii and Lalchhandama, 2020). In nematodes, it is evident that anthelmintic drugs disrupt the structural integrity of the cuticle primarily by targeting cuticular proteins, which serves as their main mechanism of action (Page et al., 2014). Martínez-Ortíz-de-Montellano et al. (2013) reported that alterations occur in adult Haemonchus sp. worms following in vitro exposure to tannin-rich extracts. In contrast to the control worms, which exhibited a smooth cuticle surface, the parasites treated with both tannin-rich plant extracts displayed significant morphological changes, including longitudinal and transverse folds as well as thicker ridges on the cuticle. These lesions were observed across the entire body of the nematodes or in patches, affecting both the cephalic region and other parts of the body, including the distal ends of the. Notably, the most pronounced changes were observed as aggregates surrounding the buccal capsule, female vulva, or anus. Barone et al. (2018) observed that the buccal region of Haemonchus sp. exposed to condensed tannins (CTs) from cranberry vines exhibited damage and the presence of aggregats. In contrast, the worms that were not subjected to exposure to these active compounds appeared normal. Tresia et al. (2016) reported that the anthelmintic activity of active compounds in plants is exerted synergistically through the damage to the cuticle and alterations in the form and permeability of the worm’s cuticle.

CONCLUSIONS AND RECOMMENDATIONS

The 7% teak leaf nanoemulsion demonstrated anthelmintic activity against Haemonchus sp. This study establishes a foundation for the use of nanoemulsions as an anthelmintic agent. However, additional in vivo studies are necessary to validate the potential application of this medicinal plant in ruminants.

ACKNOWLEDGMENTS

The authors wish to acknowledge The Ministry of Research, Technology and Higher Education of the Republic of Indonesian through Penelitian Pascasarjana Magister Program Grant Number 048/E5/PG.02.00.PL/2024. The authors wish to acknowledge Research Center for Animal Husbandry, National Research and Innovation Agency (BRIN) and Directorat of Research, Universitas Gadjah Mada.

NOVELTY STATEMENTS

The study on the anthelmintic activity of Tectona grandis leaves nanoemulsion against haemonchus in ruminants has never been done before.

AUTHOR’S CONTRIBUTIONS

Dian Indriati, Yulita Nadzifa Erista, Irkham Widiyono, Zein Ahmad Baihaqi and Yanuartono: Designed the study, collected samples, and conducted laboratory examinations; Dian Indriati: composing the initial draft. All authors contributed to drafting and revising the manuscript. All authors have read, evaluated, and given their approval for the final manuscript.

Conflict of Interest

There were no conflicts of interest reported.

REFERENCES

Ahmad N, Ahmad R, Al-Qudaihi A, Alaseel SE, Fita IZ, Khalid MS, Pottoo FH (2019). Preparation of a novel curcumin nanoemulsion by ultrasonication and its comparative effects in wound healing and the treatment of inflammation. RSC Adv., 9: 20192–20206. https://doi.org/10.1039/C9RA03102B

Amin N, Das B (2019). A review on formulation and characterization of nanoemulsion. Int. J. Curr. Pharm. Sci., 11: 1-5. https://doi.org/10.22159/ijcpr.2019v11i4.34925

Asdaq SMB, Ayeem N, Alam MT, Alaqel, SI, Imran M, Hassan EE, Rabbani SI (2022). Tectona grandis L.f: A comprehensive review on its patents, chemical constituents, and biological activites. Saudi J. Biol. Sci., 29: 1456-1464. https://doi.org/10.1016/j.sjbs.2021.11.026

Baihaqi ZA, Widiyono I, Nurcahyo W (2020). Potential of Carica pubescens fruit peel as an alternative method to control Haemonchus contortus in small ruminants. Livestock Res. Rural Dev., 32: 106.

Baihaqi ZA, Widiyono I, Angeles AA, Suwignyo B, Nurcahyo W (2023). Anthelmintic activity of Carica pubescens aqueous seed extract and its effects on rumen fermentation and methane reduction in indonesian thin-tailed sheep: An in vitro study. Vet. World, 16: 1421–1428. https://doi.org/10.14202/vetworld.2023.1421-1428

Baihaqi ZA, Sofyan A, Suwignyo B, Angeles A, Widiyono I, Nurcahyo W, Ibrahim A, Putri E, Wulandari (2024). In vivo study: The effects of Carica pubescens seed extract on the anthelmintic activity, feed digestibility, performance, and clinical parameters of thin-tailed sheep. IOP Conf. Ser. Earth and Environ. Sci., 1341:1-12. https://doi.org/10.1088/1755-1315/1341/1/012119

Balqis U, Hambal M, Rinidar, Athaillah F, Ismail, Azhar, Vanda H, Darmawi (2017). Cuticle surface damage of Ascaridia galli adult worms treated with Veitchia merrillii betel nuts extract in vitro. Vet. World, 7: 732-737. https://doi.org/10.14202/vetworld.2017.732-737

Barone CD, Zajac AM, Manzi-Smith LA, Howell AB, Reed JD, Krueger CG, Petersson KH (2018). Anthelmintic efficacy of cranberry vine extracts on ovine Haemonchus contortus. Vet. Parasitol., 253: 122–129. https://doi.org/10.1016/j.vetpar.2018.02.016

Charoensit P, Sawasdipol F, Tibkawin N, Suphrom N, Khorana N (2021). Development of natural pigments from Tectona grandis (teak) leaves: agricultural waste material from teak plantations. Sustain. Chem. Pharm., 19: 1-9. https://doi.org/10.1016/j.scp.2020.100365

Chaudhary S, Kumar S, Kumar V, Sharma R (2020). Chitosan nanoemulsions as advanced edible coatings for fruits and vegetables: Composition, fabrication and developments in last decade. Int. J. Bio. Macromol., 152: 154-170. https://doi.org/10.1016/j.ijbiomac.2020.02.276

FAO (1991). Food losses due to non-infectious and production diseases in developing countries. FAO, United Nations.

Fissiha W, Kinde MZ (2021). Anthelmintic resistance and its mechanism: A review. Infect. Drug Resist., 14: 5403–5410. https://doi.org/10.2147/IDR.S332378

Gasbarre LC, Smith LL, Lichtenfels JR, Pilitt PA (2009). The identification of cattle nematode parasites resistant to multiple classes of anthelmintics in a commercial cattle population in The US. Vet. Parasitol., 166: 281–285. https://doi.org/10.1016/j.vetpar.2009.08.018

Greiffer L, Liebau E, Herrmann FC, Spiegler V (2022). Condensed tannins act as anthelmintics by increasing the rigidity of the nematode cuticle. Sci. Rep., 12: 1–13. https://doi.org/10.1038/s41598-022-23566-2

Gururaja, Himanshu J, Ishwara B, Satyanarayana D, Shastry CS (2011). Anthelmintic activity of Tectona grandis linn fruits. Int. Res. J. Pharm., 2: 219-221.

Handayanta E, Hadi R, Barido FH (2023). Screening the anthelmintic effects of leaves and fruit extracts from various Acacia species as a potential feed for small-ruminant animals. Biodiversitas, 24: 3140-3144. https://doi.org/10.13057/biodiv/d240606

Hikmawanti NP, Fatmawati S, Asri AW (2020). The effect of ethanol concentrations as the extraction solvent on antioxidant activity of katuk (Sauropus androgynus (L.) Merr.) leaves extracts. IOP Conf. Ser. Earth Environ. Sci., 755. https://doi.org/10.1088/1755-1315/755/1/012060

Hrckova G, Velebny S (2013). Pharmacological potential of selected natural compounds in the control of parasitic disease. Springer Wien Heidelberg New York Dordrecht London, 1325-1327.

Hou B, Yong R, Wuen J, Zhang Y, Buyin B, Subu D, Zha H, Li H, Hasi S (2022). Positivity rate investigation and anthelmintic resistance analysis of gastrointestinal nematodes in sheep and cattle in Ordos, China. Animals, 12(7). https://doi.org/10.3390/ani12070891

Javalgikar A, Shaikh H, Sargar M, Survanshi H, Rathod M (2019). In vitro anti-inflamatory and anthelmintic activity of Tectona grandis leaves extract. Int. J. Herb. Med., 7: 36-40.

Koffi EN, Meudec E, Adje FA, Lozand PR, Lozand YF, Bekroa YA (2015). Effect of reserve osmosis concentration coupled with drying processes on polyphenols and antioxidant activity obtained from Tectona grandis leaf aqueous extracts. J. Appl. Res. Med. Aromat. Plants, 2: 54-59. https://doi.org/10.1016/j.jarmap.2015.03.001

Lalthanpuii PB, Lalchhandama K (2020). Scanning Electron Microscopic Study of the anthelmintic effects of some anthelmintic drugs on poultry nematode, Ascaridia galli. Adv. Anim. Vet., 8: 788-793. https://doi.org/10.17582/journal.aavs/2020/8.8.788.793

Lisnanti EF, Lokapirnasari WP, Hestianah EP, Al Arif MA, Baihaqi ZA (2023). The Effectiveness of Giving Marsh Fleabane (Pluchea indica L.) Water Extract On Broiler Hematology and Blood Glucose. Adv. Anim. Vet., 11: 1348-1356. https://doi.org/10.17582/journal.aavs/2023/11.8.1348.1356

Lokapirnasari WP, Al-Arif MA, Hidayatik N, Safiranisa A, Arumdani DF, Zahirah AI, Yulianto AB, Lamid M, Marbun TD, Lisnanti EF, Baihaqi ZA, Khairullah AR, Kurniawan SC, Pelawi EBS, Hasib A (2024). Effect of probiotics and acidifiers on feed intake, egg mass, production performance, and egg yolk chemical composition in late-laying quails. Vet. World, 17: 462–469. https://doi.org/10.14202/vetworld.2024.462-469

Macedo F, Marsico ET, Conte-Júnior CA, Furtado LA, Brasil TF, Pereira NAD (2015). Short communication: Macrocyclic lactone residues in butter from Brazilian markets. J. Dairy Sci., 98: 3695–3700. https://doi.org/10.3168/jds.2014-9130

Maestrini M, Tava A, Mancini S, Salari F, Perrucci S (2019). In vitro anthelmintic activity of saponins derived from Medicago spp. plants against donkey gastrointestinal nematodes. Vet. Sci., 6: 1-8. https://doi.org/10.3390/vetsci6020035

Markham KR (1988). Distribution of flavonoids in the lower plants and its evolutionary significance in the flavonoids. Springer, 427-468. https://doi.org/10.1007/978-1-4899-2913-6_12

Martínez-Ortíz-de-Montellano C, Arroyo-López C, Fourquaux I, Torres-Acosta JFJ, Sandoval-Castro CA, Hoste H (2013). Scanning electron microscopy of Haemonchus contortus exposed to tannin-rich plants under in vivo and in vitro conditions. Exp. Parasitol., 133(3): 281–286. https://doi.org/10.1016/j.exppara.2012.11.024

Miro MV, Luque S, Cardozo P, Lloberas M, Sousa DM, Soares AMS, Costa-Junior LM, Virkel GL, Lifschitz L (2020). Plant-derived compounds as tool for the control of gastrointestinal nematodes: modulation of abamectin pharmacological action by carvone. Front. Vet. Sci, 7:601750. https://doi.org/10.3389/fvets.2020.601750

Mondal H, Hossain H, Awang K, Saha S, Rashid SMU, Islam MK, Rahman MS, Jahan IA, Rahman MM, Shilpi JA (2015). Anthelmintic activ­ity of ellagic acid, a major constituent of Alternanthera sessilis against Haemonchus contortus. Pak. Vet. J., 35: 58–62.

Mukhriani (2014). Ekstraksi, pemisahan senyawa, dan identifikasi senyawa aktif. Jurnal Kesehatan, 7 (2).

Mumed HS, Nigussie DR, Musa KS, Demissie AA (2022). In vitro anthelmintic activity and phytochemical screening of crude extracts of three medicinal plants against Haemonchus contortus in sheep at Haramaya Municipal abbatoir, Eastern Hararghe. J. Parasitol. Res., 6331740. https://doi.org/10.1155/2022/6331740

Murukan G, Kumara M (2018). Comparison of phenolic acids and antioxidant activities of young and mature leaves of Tectona grandis. Asian J. Pharm, 11: 60. https://doi.org/10.22159/ajpcr.2018.v11i1.19430

Ogunmefun OT, Ekundayo EA, Akharaiyi FC, Ewhenodere D (2017). Phytochemical screening and antibacterial activities of Tectona grandis L.f (teak) leaves on microorganisms isolated from decayed food samples. Trop. Plant Res., 4: 376–382. https://doi.org/10.22271/tpr.2017.v4.i3.049

Page AP, Stepek G, Winter AD, Pertab D (2014). Enzymology of the nematode cuticle: A potential drug target. Int. J. Parasitol, 4: 133–141. https://doi.org/10.1016/j.ijpddr.2014.05.003

Prasetyo EN, Rokana E, Baihaqi ZA, Samudi S (2024). Anthelmintic effects of podang mango (Mangifera Indica) fruit peel waste extract through in vivo application on Indonesian Etawa goat production and health. Vet. World, 17: 1291–1298. https://doi.org/10.14202/vetworld.2024.1291-1298

Prayudi SKA, Effendi MH, Lukiswanto BS, Az Zah-Ra RL, Benjamin MI, Kurniawan SC, Khairullah AR, Silaen OSM, Lisnanti EF, Baihaqi ZA, Widodo A, Riwu KHP (2023). Detection of genes on Escherichia coli producing extended spectrum β-lactamase isolated from the small intestine of ducks in traditional markets Surabaya city, Indonesia. J. Adv. Vet. Res., 13: 1600-1608.

Puspitasari D, Rahmawati N, Putri NK, Pradipta MP (2022). Nanoemulsi ekstrak wortel dan virgin coconut oil sebagai suplemen pro- vitamin a untuk mencegah kekurangan vitamin a. Agritech, 42: 65–74. https://doi.org/10.22146/agritech.47743

Rokana E, Fatimah IR, Dianingtyas BD, Hasanah N, Wulandari, Baihaqi ZA (2024). Impact of various fiber sources in ration formulas on feedlot performance of sheep in Indonesia. J. Anim. Health. Prod., 12: 325-330. https://doi.org/10.17582/journal.jahp/2024/12.3.325.330

Sakti AA, Baihaqi ZA, Suwignyo B, Sofyan A, Herdian H, Kustantinah (2024). Anthelmintic activity of red macroalgae Acrocystis sp. and Acanthophora sp. etanolic extract against Haemonchus contortus in sheep in vitro. IOP Conf. Ser.: Earth Environ. Sci., 1360: 012004. https://doi.org/10.1088/1755-1315/1360/1/012004

Sa’adah H, Nurhasnawati H (2017). Perbandingan pelarut etanol dan air pada pembuatan ekstrak umbi bawang tiwai (Eleutherine americana Merr) menggunakan metode maserasi. Jurnal Ilmiah Manuntung, 1:149–153. https://doi.org/10.51352/jim.v1i2.27

Sadeq ZA (2020). Review on nanoemulsion: preparation and evaluation. Int. J. Drug Deliv. Technol, 10: 187-189. https://doi.org/10.25258/ijddt.10.1.33

Sreeja MK, Poulose PA (2017). Comparative study of anthelmintic activity of albendazole nano emulsion containing oregano essential oil. Int J Recent Sci Res, 8: 20111-20119.

Toklo PM, Ladeken EY, Linden A, Hounzangbe-Adote S, Kouam SF, Gbenou JD (2021). Anthelmintic flavonoids and other compounds from Combretom glutinosum perr. ex dc (Combretaceae) leaves. Acta Cryst., C77: 505–512. https://doi.org/10.1107/S2053229621007841

Tresia GE, Evvyernie D, Tiuria R (2016). Phytochemical screening and in vitro ovicidal, larvacidal, and nematicidal effects of Murraya paniculata (L.) jack extract on gastrointestinal parasites of goats. Media Peternakan, 39: 173–179. https://doi.org/10.5398/medpet.2016.39.3.173

Vatsraj S, Chauhan K, Pathak H (2014). Formulation of a novel nanoemulsion system for enhanced solubility of a sparingly water soluble antibiotic, clarithromycin. J. Nanosci., 1–7. https://doi.org/10.1155/2014/268293

Victor S, Ouwor B, Yusuf A (2014). In-vitro anthelmintic bioactivity study of Eclipta prostrata L. (whole plant) using adult Haemonchus contortus worms a case study of Migori country, Kenya. IOSR J. Pharm. Biol. Sci., 9(6): 45–53. https://doi.org/10.9790/3008-09644553

Yanuartono, Indarjulianto S, Nururrozi A, Raharjo S, Purnamaningsih, H (2020). Penggunaan antiparasit ivermectin pada ternak: antara manfaat dan risiko. Jurnal Sain Peternakan Indonesia, 15: 110–123. https://doi.org/10.31186/jspi.id.15.1.110-123