Cystic echinococcosis (CE), a zoonotic parasitic disease, is caused by the metacestode of the canine larval tapeworm Echinococcus granulosus. The infection results from the development of hydatid cysts, especially in the liver and lungs, although cysts can form in any part of the body (Budke et al., 2009). CE infestation results in economic losses, including carcass condemnation, reduced milk production and fertility, as well as increased weight loss and mortality (Bekele & Butako, 2011; Jairus et al., 2023). Economic losses include medical and veterinary costs, which have been estimated at around $3 billion a year (WHO, 2024).
Currently, parasitic diseases are controlled by synthetic drugs, which have signifi cant negative impacts such as their high cost, the potential for drug resistance and their environmental impact. As a result, new therapies, such as those using traditional medicinal plants, are becoming increasingly popular (Kheirandish et al., 2020). However, these medicinal plants require further study regarding their characteristics, safety, and effects (Nascimento et al., 2000). In this context, researchers are currently seeking new, safer, and more effective protoscolicidal agents for the treatment of CE (Alvi et al., 2022).
Herbal medicinal products such as Olea europaea (Zibaei et al., 2012), Berberis vulgaris (Rouhani et al., 2013), Zingiber officinale (Amri & Touil-Boukoffa, 2016), Blepharocalyx salicifolius (Noal et al., 2017), Artemisia sieberi (Vakili et al., 2019), Punica granatum (Labsi et al., 2019), Atriplex halimus (Bouaziz et al., 2021), Malva sylvestris (Jasim, 2023), Juniperus phoenicea (Al-khlifeh et al., 2023), Calotropis procera (Al-khlifeh et al., 2023), and Artemisia judaica (Al-khlifeh et al., 2023) have shown promising effects on killing protoscoleces in vivo and/or in vitro.
Hypericum perforatum is a plant widely used in traditional medicine, and its antimicrobial properties have been demonstrated. (Kladar et al., 2017; Sherif et al., 2023). Thymus vulgaris possesses several medicinal properties, including anticoccidial (Jamroz et al., 2003) and anthelmintic (Rasooli et al., 2006) effects. It contains many substances with therapeutic value, such as thymol, eugenol, flavonoids, and others (Amarowicz et al., 2008). Pimenta racemose has traditionally been used for medicinal purposes, and its antibacterial effect has previously been shown (Deberdt et al., 2018). A study conducted on the antimicrobial activity of Mentha piperita essential oils showed that it has the potential to control several pathogens such as fungi and bacteria (Camele et al., 2021).
The aqueous extraction method is a valuable traditional practice. It provides a safe and effective way of extracting medicinal compounds from plants. This technique can be very efficient for the extraction of water-soluble phytochemicals, such as flavonoids, tannins, and alkaloids (Sarikurkcu et al., 2020).
In this present study, we evaluated the in vitro protoscolicidal effect of Hypericum perforatum, Thymus vulgaris, Pimenta racemosa, and Mentha piperita against Echinococcus granulosus protoscoleces using the aqueous extraction method.
Cysts were collected from the livers and lungs of locally infected sheep that had been slaughtered in a private operator’s slaughterhouse in Van, Turkey. The hydatid fluid was poured into a small beaker and left to settle for 30 min to allow the protoscoleces to precipitate. The upper layer was gently removed, and the precipitated protoscoleces were rinsed three times in 0.9 % sodium chloride. The viability of the protoscoleces was confirmed by a 0.1 % eosin exclusion test under the light microscope (Leica DM500).
Plants were obtained from the seller of medicinal herbs in the center of Van province, Turkiye. Plant parts were washed carefully and then air-dried. Once air-dried, the samples were pulverized in a mortar, and 40 g of each crumbled sample was poured into a glass beaker containing 160 mL of dH2O. Samples were incubated at room temperature for 72 h. Then, the filtration process was performed using glass filter funnels and filter papers. The filtered samples were evaporated for the condensation process and dried in sterilizator (Termal Laboratory, Type: G11420SD, Holland) at 45 °C. The samples were then stored at 4°C for later processing.
Three concentrations, 50, 100, and 150 mg/mL, were prepared by adding 1.5, 3, and 4.5 g of the final plant product, respectively, and dissolving them in 30 mL PBS buffer in tubes. Protoscoleces were mixed with the prepared herbal extract at the desired concentration in a test tube and incubated for the specified time. The three concentrations of herbal extracts were tested for 5, 10, and 60 minutes against E. granulosus protoscoleces, and their protoscolicidal effect was evaluated using the eosin (0.1 %) exclusion test. A total of 150 E. granulosus protoscoleces were counted under the light microscope, and the number of viable and dead protoscoleces was immediately recorded. All tests were performed in triplicate, and the mean value was taken as the result. Hypertonic saline (20 %) was used as the positive control, and sodium chloride (0.9 %) was used as the negative control. Previous studies describing the active molecules in the aqueous extracts of the plant samples are mentioned in the discussion section, along with their corresponding references.
Statistical analysis was carried out using SPSS software (version 21). Differences between the experimental and control groups were analyzed using one-way ANOVA with Tukey post-hoc tests. Data are represented as mean ± standard deviation (SD). Furthermore, P<0.001 was considered statistically significant.
This article does not contain any studies with human participants or animals.
In vitro protoscolicidal effects of 50, 100, and 150 mg/mL of herbal extracts were demonstrated after 5, 10, and 60 min of incubation (Table 1). Mortality rates on protoscoleces for 50, 100 and 150 mg/ml concentrations of H. perforatum, T. vulgaris and P. racemosa extracts following 10 min were 49.1 ± 1.35, 52.2 ± 2.7, 54.4 ± 1.35; 40.2 ± 2.19, 66.2 ± 3.64, 98 ± 1.15; and 52.2 ± 2.92, 76.9 ± 2.99, 96.8 ± 3.88, respectively. Mortality rates of T. vulgaris and P. racemosa at 100 mg/ml concentrations following 5 minutes were 60.9 ± 2.25 and 55.1 ± 3.95, respectively. Mortality rates of T. vulgaris and P. racemosa at 150 mg/ml concentrations following 5 minutes were 96.5 ± 2.58 and 60.2 ± 2.73, respectively. Mortality rates on protoscoleces for 50, 100 and 150 mg/ml concentrations of Hypericum perforatum, Thymus vulgaris and Pimenta racemosa extracts following 60 min were 88.9 ± 3.85, 91.6 ± 3.7, 93.3 ± 3.29; 56.9 ± 1.95, 99.3 ± 0.39, 99.8 ± 0.2; and 95.3 ± 2.31, 96.7 ± 1.76, 98.2 ± 0.97, respectively. The combined effect of higher concentration and longer exposure time leads to significantly higher mortality rates. These results showed that all three plant extracts had a statistically significant effect on protoscoleces mortality (P<0.001) when compared with the negative control. Mentha piperita extract showed significantly lower mortality rates compared to other herbal extracts, even at higher concentrations and longer exposure times. In vitro protoscolicidal activity of M. piperita extract was limited to approximately 20 % at the highest dose and exposure time. The T. vulgaris extract showed a more substantial effect on mortality rates compared to other herbal extracts, especially at higher concentrations and longer exposure times. The scolicidal effect of a 150 mg/mL T. vulgaris extract could be observed at one-minute intervals (Fig. 1). The positive and negative controls validated the reliability of the results.
Effect of T. vulgaris extract (150 mg/ml) on protoscoleces. The same microscopic image was photographed at one-minute intervals to observe how the protoscoleces took up the 0.1% eosin after their death (A: min 1, B: min 2, C: min 3, D: min 4, E: min 5, and magnification: 40X).
In vitro protoscolicidal activity of different concentrations of herbal extracts at 5, 10, and 60 min.
| Experimental Groups | Samples | Concentration | Mortality rate (%) with following exp. time | ||
|---|---|---|---|---|---|
| 5 min | 10 min | 60 min | |||
| Treatment with herbal extract | H. perforatum (Leaves and flowers) | 50 mg/ml | 36.4 ± 1.74 | 49.1 ± 1.35 | 88.9 ± 3.85 |
| T. vulgaris (Leaves) | 50 mg/ml | 33.7 ± 1.56 | 40.2 ± 2.19 | 56.9 ± 1.95 | |
| P. racemose (Leaves) | 50 mg/ml | 44.6 ± 3.53 | 52.2 ± 2.92 | 95.3 ± 2.31 | |
| M. piperita (Leaves) | 50 mg/ml | 10.44 ± 0.97 | 11.6 ± 1.98 | 14.2 ± 2.73 | |
| Positive Control | NaCl | 20 % | 100 ± 0 | 100 ± 0 | 100 ± 0 |
| Negative Control | NaCl | 0.90 % | 2.2 ± 0.46 | 3.3 ± 0.53 | 4.5 ± 0.97 |
Herbal medicines have long played a central role in complementary and alternative medicine worldwide (Kohansal et al., 2017). Medicinal plants, due to their minimal side effects, low cost, and wide availability, have become a source of new drugs for treating various diseases. There has been an increasing amount of research into herbal therapies and their beneficial effects against parasitic diseases. Nevertheless, it should not be ignored that the global trend in research is about to change, as the focus shifts from growing plants with medicinal properties to finding new drugs (Salmerón-Manzano et al., 2020).
Current treatments of CE include surgery, chemotherapy and percutaneous aspiration-injection-reaspiration. Moreover, albendazole and mebendazole are used as oral anthelmintics (Adas et al., 2010). However, there is often no complete recovery after treatment, and 40 % of patients do not achieve a sufficient response (Naguleswaran et al., 2006; Nicolao et al., 2014). Due to side effects, drug resistance and relapse of Echinococcus granulosus infestation following traditional therapies, the search for innovative therapeutic approaches, such as natural products, seems necessary (Boakye et al., 2023). It is therefore of vital importance to develop novel, safe, and efficient scolicidal agents.
A review of herbal medicines against hydatid disease revealed that methanolic extraction was the most commonly used extraction method in studies conducted between 2000 and 2021 (Alvi et al., 2022). This was followed by hydrodistillation and extraction methods using ethanolic and aqueous solvents, respectively. The ethanolic extract method had a stronger and faster scolicidal activity against E. granulosus protoscoleces compared to the aqueous extract. (Benmarce et al., 2024). However, aqueous extraction is a simple, environmentally friendly, safe and cost-effective method of extracting (El-Desouky, 2021).
It appears that there are very few in vivo studies on the effectiveness of medicinal plants against protoscoleces of E. granulosus. (Alvi et al., 2022). Protoscolicidal effects of Punica granatum’s peels were tested using an aqueous extraction method on mice (Labsi et al., 2016). A total of 16 mg/mL of plant extracts was applied for two days. Scolicidal efficacy was found to be 100 %. In a similar study conducted on mice, extracts from Punica granatum peels were used at a concentration of 0.65 mg/ml (Labsi et al., 2019). Plant extracts were applied for 60 days, and scolicidal efficacy was found to be 66.7 %. Atriplex halimus leaves were used against protoscoleces using the aqueous extract method, and mortality rates were 99.36 % and 100 % at 60 and 100 mg/ml, respectively, after 120 min exposure (Bouaziz et ark, 2021). The extracts had no cytotoxic effect on murine peritoneal macrophages. Several studies have investigated the in vitro efficacy of medicinal plants in treating E. granulosus protoscoleces using the aqueous extract method. Olea europaea leaf extracts were tested against the protoscoleces, resulting in a mortality rate of 96.7 % at 1 mg/mL after 120 minutes of incubation (Zibaei et al., 2012). Berberis vulgaris fruit extracts were used against the protoscoleces, and a 100 % mortality rate was observed at 4 mg/ml after 30 minutes of incubation (Rouhani et al., 2013). These results demonstrate that the scolicidal activity was highly effective at low concentrations and short exposure times. Zingiber officinale exhibited a time-dependent protoscolicidal effect at 24 h and 48 h, with maximum reductions of 89.72 % and 100 % (P < 0.0001) at 100 μg/ml, respectively (Amri & Touil-Boukoffa, 2016). Blepharocalyx salicifolius leaf extract (gallic acid) showed 100 % scolicidal activity against Echinococcus ortleppi at a concentration of 200 mg/ml for 5 min. (Noal et al., 2017). Artemisia sieberi extract showed 92.6 % ± 1.28 scolicidal activity against E. granulosus protoscoleces at a concentration of 75 mg/ml for 10 min (Vakili et al., 2019). The alcoholic and aqueous extracts of Malva sylvestris leaves had a 100 % protoscolocidal effect on E. granulosus protoscoleces at a concentration of 60 mg/ml for 60 min (Jasim, 2023). The anthelmintic effect of the essential oil of Thymus vulgaris against protoscoleces and cysts of E. granulosus has been previously shown (Pensel et al., 2014). In the present study, a total of four plants were examined for their protoscolocidal efficacy against the protoscoleces of E. granulosus (Table 1). The aqueous extracts of Hypericum perforatum, Thymus vulgaris, and Pimenta racemose had a significant effect on protoscolex mortality (P<0.001). The extract of T. vulgaris showed a more substantial effect on mortality rates compared to other groups, achieving near-complete mortality (up to 99.8 %) at higher concentrations and longer exposure times. Moreover, we thought it would be noteworthy to present photographically how rapidly the scolicidal effect of 150 mg/mL T. vulgaris extract occurs at one-minute intervals (Fig. 1).
A study conducted by Maggiore et al. (2012) showed a time-dependent anthelmintic activity of Mentha spp—essential oils on E. granulosus protoscoleces and metacestodes. The hydrodistillation extraction method was used in the study. Mentha pulegium had a much stronger anthelmintic efficacy than M. piperita. The scolicidal effect of M. piperita was slower, with nearly 50 % efficacy after 24 days. In our study, the in vitro protoscolicidal effect of M. piperita (150 mg/ml) was approximately 20 % in one hour. Considering the current and previous studies cited in the paper, it is clear that the scolicidal effect is dependent on both duration and dose in most cases.
Hypericum perforatum is characterized by the presence of several key bioactive compounds, including hyperoside (Chen et al., 2018), hesperidin (Hernández-Saavedra et al., 2016), quercetin (Chen et al., 2018), and chlorogenic acid (Mohammed et al., 2019), which are identified as its primary constituents. A study by Sarikurkcu et al. (2020) showed that the aqueous extract of H. perforatum contained a total of 26 phytochemical compounds, with hyperoside, hesperidin, quercetin, and chlorogenic acid as the major constituents, as determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. Thymus vulgaris, commonly known as thyme, is widely used for various medicinal applications. The essential oil of thyme is its active component, with thymol and carvacrol serving as the main compounds (Gavarić et al., 2015). The essential oil of Pimenta racemosa was extracted from the aerial parts by hydrodistillation and analysed by GC/MS. Forty-five compounds were identified, of which eugenol, β-pinene, linalool and limonene were the major components (Elshaarawy et al., 2024). Aqueous extracts of M. piperita leaves contain flavonoids such as eriocitrin, luteolin-7-O-glucoside and rosmarinic acid. While the highest amounts can be obtained with methanolic and acetonitrile extraction methods, low amounts can be obtained with the aqueous extraction method (Hudz et al., 2023). We believe that these bioactive compounds in each of the herbal extracts, either individually or in combination, are responsible for killing the parasite in our study. The current study only suggests the potential efficacy of the aqueous extracts. However, without in vivo (animal or clinical) studies, the actual therapeutic effectiveness remains uncertain.
In conclusion, our findings suggest that the aqueous extracts of Hypericum perforatum, Thymus vulgaris, and Pimenta racemosa could be a potential source of protoscolicidal agents, making them promising candidates for further studies. Additional in vitro and in vivo studies are needed to comprehensively evaluate the anthelmintic constituents of these plants for the therapy of cystic echinococcosis. Future studies may lead to exploring the mechanisms behind the herbal extract’s effectiveness and investigating compounds with enhanced therapeutic value.