Evaluation of anthelminthic and antiprotozoal activity of myrrh (Commiphora myrrha) methanolic extract

Parasitic infections caused by protozoans and helminths induce considerable health problems in various animal species (Mehlhorn, 2014). Helminth infections are among the most common form of gastrointestinal parasites in birds that leads to economic losses (Newbold et al., 2017; Al-Quraishy et al., 2020). Weakness is considered a major complaint of helminth infections resulting from malnutrition, anemia, and eosinophilia (Jones & Berkley, 2014). Anthelminthic drugs are used for expelling parasitic worms from the body, however, they induce side effects, especially for host tissue (Hong, 2018). Coccidiosis is a protozoal disease caused by Eimeria species (Kommu et al., 2016). Eimeria labbeana-like is a coccidian parasite that was first reported in domesticated pigeons (Yang et al., 2016). Infection of pigeons with coccidian parasites causes changes in physical appearance (Sood et al., 2018). Coccidiosis is mostly treated with synthetic anticoccidial drugs, but this approach is facing a serious threat of the development of resistance in Eimeria strains (Grandi et al., 2016).

To control coccidiosis and helminthiasis in various animal species, different alternative options and protocols were effectively used worldwide (Liaqat et al., 2016). The use of herbs as medicine is becoming increasingly common, either as home remedies or as complementary and alternative medicines (Satyavati, 1990). Plant therapy is frequently considered to be less toxic with the least side effects than synthetic ones. The plant-derived medicines are based on the premise that they contain natural substances that can promote health and alleviate disease status (Swayamjot et al., 2005).

Myrrh, as traditional natural medicine, is an aromatic gum resin, which was the plant stem resinous exudate of Commiphora myrrha which belongs to the family Burseraceae (Alyafei, 2020). Myrrh is widely used as a home remedy in Saudi Arabia (Al-Faris et al., 2008; Bakhotmah & Alzahrani, 2010). Chemical compounds present in myrrh resins include triterpenoids, diterpenoids, steroids, and lignans (Hanus et al., 2005; Tonkal & Morsy, 2008). Myrrh has been shown to have a wide variety of therapeutic uses as an antimicrobial (Romero et al., 2005; Rahman & Gibbons, 2007; Alzahrani et al., 2011; Kuete et al., 2012), anticancer (Shoemaker et al., 2005), anti-inflammatory (Qureshi et al., 1993; Akbar, 2020), lipid-lowering (Omer & Al-Dogmi, 2018), antirheumatic (Su et al., 2015), antioxidant (El-Ashmawy et al., 2006; Ashry et al., 2010), and hypotensive (Abdul-Ghani & Amin, 1997). It is also known to stimulate uterine tone and promote uterine blood flow (Michie & Cooper, 1991). Moreover, it is believed to be effective in treating various parasites including Eimeria stiedae (Baghdadi & Almathal, 2010), Trichinella spiralis (Basyoni & El-Sabaa, 2013; Attia et al., 2015; Abd-Elrahman et al., 2020; Abuelenain et al., 2021, 2022), Fasciola gigantica (Massoud et al., 2013), and Schistosoma mansoni (Osman et al., 2010).

This study aims to evaluate the potential role of Commiphora myrrha extract as an anthelminthic and anticoccidial effector against E. labbeana-like.

This research was approved by the Research Ethics Committee (REC) at King Saud University (approval number KSU-SU-23-45).

Fig. 3.

FT-IR of MyE showed major bands for the twelve compounds at 3423 cm⁻¹, 2969.17 cm⁻¹, 2932.49 cm⁻¹, 1739.07 cm⁻¹, 1614.29 cm⁻¹, 1438.45 cm⁻¹, 1381.21 cm⁻¹, 1245.04 cm⁻¹, 1039.34 cm⁻¹, 766.62 cm⁻¹, 729.36 cm⁻¹, and 597.88 cm⁻¹ (Fig. 1 and Table 1). O-H stretching was indicated by the band at 3423 cm⁻¹ confirming the presence of an alcohol. The bands at 2969.17 and 2932.49 cm⁻¹ implied N-H stretching for the presence of amine salt. C-O stretching at 1739.07 cm⁻¹ confirms the presence of esters. The band at 1614.29 cm⁻¹ corresponds to C=C stretching for the presence of the α,β-un-saturated ketone. The band 1438.45 cm⁻¹ (O-H bending), 1381.21 cm⁻¹ (S=O stretching), 1245.04 cm⁻¹ (C-N stretching), 1039.34 cm⁻¹ (S=O stretching), 766.62 cm⁻¹ (C-CI stretching), 729.36 cm⁻¹ (C=C bending), and 597.88 cm⁻¹ (C-I stretching) assigned to a carboxylic acid, sulfonyl chloride, amine, sulfoxide, halo compound, and alkene, respectively (Table 1).

Fig. 4.

Fig. 5.

MyE displays a relatively comparable anthelmintic activity with reference standard mebendazole against the adult E. fetida worms. Paralysis and death time of the worms were recorded, and the obtained results are shown in Figures 2 and 3. This experiment was carried out for up to 48 minutes. There was no paralysis recorded in the dist. H₂O (control group). From the observations achieved, a higher concentration of MyE (100 mg/ml) showed a paralytic effect much earlier (7.88 ± 0.37 min) and the time to death was shorter (9.24 ± 0.60 min) for almost all the worms. Mebendazole at 10 mg/ml showed paralysis and death times after 13.91 ± 0.37 and 18.20 ± 3.98 min, respectively. The other MyE concentrations showed a marked degree of anthelmintic activity.

Microscopic examination revealed uniform normal body architecture for E. fetida worms in water (Fig. 4, Supplementary Fig. 1). On the other hand, all E. fetida worms exposed to MyE had alterations in the topography including a decrease in the length of body segments accompanied by cuticular thickness (Fig. 4, Supplementary Fig. 1). All E. fetida worms treated with mebendazole showed observable destruction of the cuticle layer (Fig. 4, Supplementary Fig. 1).

Oocyst incubation with K₂Cr₂O₇ (2.5 %), MyE (100, 50, and 25 mg/ml), amprolium, phenol, and Dettol^TM showed different levels of sporulation (Fig. 5). The lowest rate of sporulation recorded for the higher concentration (100 mg/ml) of MyE is 5.23 % (at 24 hr) and 10.65 (at 36 hr). After incubation with formalin, the unsporulated E. labbeana-like oocysts showed no rate of sporulation. Incubation with MyE (100 mg/ml) for 24 and 36 hr inhibited oocysts sporulation by 90.95 and 87.17 %, respectively. MyE (50 and 25 mg/ml), amprolium, Dettol^TM, and phenol induced variable inhibition levels at 36 hr of 40.17 %, 29.34 %, 45.09 %, 85.11 %, and 61.58 %, respectively (Fig. 6).

In our environment, there are different pathogens affecting various animal species causing parasitic diseases (including coccidiosis and helminthiasis) that lead to severe economic losses. Many therapeutic agents are available to control and management of these diseases, but these agents are also now adopting serious side effects and development of resistance and therefore, are no more effective in the management of infections (Chartier et al., 2001). These factors paved the way for herbal remedies as alternative agents (Coles, 1997). This study aimed to evaluate the effectiveness of one of the most famous herbal remedies in Saudi Arabia, myrrh, as an anthelmintic and anti-coccidial effector.

Fig. 6.

Anthelmintic treatments are known to act by causing irritation resulting in restriction of movement and further leading to paralysis and/or death of worms (Mackenstedt et al., 1993; Kopp et al., 2008; Lalthanpuii & Lalchandama, 2020). In this study, in earthworms E. fetida, the methanolic extract of MyE showed anthelmintic activity in a dose-dependent manner. The activity of MyE at 100 mg/ml was found to be inversely proportional to the time taken for paralysis/death of the earthworms. This result agreed with previous studies which reported that the presence of many classes of phytoconstituents of myrrh, especially terpenoids, propose an endogenous action via interaction with the polysaccharides of the worm cuticle and leads to paralysis and death in the worm (Hanus et al., 2005; Tonkal & Morsy, 2008). Borgesa and de Borgesa (2016) showed that terpenoids have antiparasitic properties since they disrupt the fluidity and permeability of the membrane of the parasite.

Histopathology has validated the in vitro study and examined the topographical effects of MyE in comparison to the standard drug on the worms to assess anthelmintic activity. The cuticular surface of the worms treated with MyE showed extraordinary modifications, including significant shrinking. This agreed with Abu Hawsah et al. (2023) who described how anthelmintic treatments caused modifications to the worm’s body surfaces. However, mebendazole has been shown to affect worms by destroying the cytoskeletal structure of the worm thereby causing paralysis, which agreed with the previous study of Kern (2003) that mebendazole is known to block microtubule functions of parasites through inhibition of polymerization of β-tubulin and glucose uptake which eventually lead parasites to be in shortage of glycogen. Wang (2010) stated also that therapeutic drugs have been reported to affect the permeability of the cell membrane of worms, causing vacuolization and disintegration of the upmost layer.

Coccidiostats are a group of analogs to thiamine (vitamin B1) that act by inhibiting the uptake of thiamine which is required for many essential metabolic reactions for the Eimeria parasites (Kart & Bilgili, 2008). The excessive use and misuse of these drugs have led to the emergence of drug-resistant strains of Eimeria species (Noack et al., 2019). In this study, amprolium has been reported to inhibit sporulation at 36 hr by 45.09 %. As a result, developing new drugs from medicinal plants is a potentially sustainable alternative to conventional chemical agents. In this study, MyE exhibited anticoccidial effect in the in vitro experiment by inhibiting sporulation of E. labbeana-like oocysts in a dose-dependent manner, which is attributable to phytoconstituents studied by Mohamed et al. (2014), Ahamad et al. (2017), Alasady et al. (2021), Koriem (2022) which interrupt the metabolism of Eimeria parasites. This finding agreed with the data presented by Baghdadi and Almathal (2010) and Massoud et al. (2010) for the efficacy of myrrh in controlling coccidiosis. In this study, phenol and Dettol^TM have been shown significant degrees of sporulation inhibition at 36 hr reached to 85.11 %, and 61.58 %, respectively. These compounds act as chemical substances that elevate the impermeability of the oocyst wall to water-soluble substances and become more resistant to proteolysis, which agreed with Gadelhaq et al. (2018), Thagfan et al. (2020), and Al-Otaibi et al. (2023). Moreover, 5 % formalin showed 100 % sporulation inhibition of E. labbeana-like oocysts, which agreed with Kasem et al. (2019), Felici et al. (2021), and Abu Hawsah et al. (2023) reported that this highly reactive chemical interacts with proteins of the oocyst wall in vitro and inhibits oocysts sporulation.

The findings of this study have shown promising anthelmintic and anticoccidial activities suggesting the possible use of myrrh in intestinal parasite control. Future studies are needed to know the mechanism of myrrh’s action on both parasite and the host tissues, as well as further fractionation of the herb to many molecules and to select the most potent one for the antiparasitic effect.