Assessment of anthelmintic activity of Artemisia judaica methanol extracts against Eisenia fetida: in vitro investigationABSTRACT
Artemisia species are known for their abundance in sesquiterpene lactones and antioxidant compounds like flavonoids and phenolic acids, offering potential health benefits for both humans and animals. This study aimed to evaluate the phytochemical profiling, cytotoxic, and anthelmintic activity of Artemisia judaica leaves methanolic extracts (AJLE). AJLE were produced and tested in vitro for anthelmintic action against Eisenia fetida. The study utilized different concentrations of AJLE extract, 25, 50, and 100 mg/mL, Albendazole (10mg/mL) was used as a positive control, and distilled water served as a negative control. Cytotoxicity analysis for the extract at different doses (µg/mL) against colon (HCT116) and liver (Huh-7) cell lines after 48 hours of incubation was conducted using an MTT assay. The methanolic extract was analyzed for its phytochemical composition using GC-MS equipment. The GC-MS spectrum has identified 19 different biomolecules. Regarding anthelminthic activity, the most effective dose of AJLE (100 mg/mL) resulted in paralysis and death within 7.502±0.812 and 8.190±0.554 minutes, respectively. In comparison, Mebendazole showed lower efficiency, resulting in death and paralysis at 18.2±0.980 and 13.91±0.373 minutes, respectively. Histological analysis of treated worms revealed significant surface structure anomalies. Furthermore, AJLE exhibited moderate cytotoxic effects against HCT116 and Huh-7 cell lines, with IC50 values of 287.55±2.89μg/ml and 324.2±2.9μg/ml, respectively. This study discovered that AJLE is a rich source of bioactive ingredients and can be used to treat helminthiasis infection.
Keywords:
Artemisia judaica; cytotoxicity; anthelmintic; E. fetida
RESUMO
As espécies de Artemisia são conhecidas por sua abundância em lactonas sesquiterpênicas e compostos antioxidantes como flavonoides e ácidos fenólicos, oferecendo possíveis benefícios à saúde de humanos e animais. O objetivo deste estudo foi avaliar o perfil fitoquímico, a atividade citotóxica e anti-helmíntica dos extratos metanólicos das folhas de Artemisia judaica (AJLE). Os AJLE foram produzidos e testados in vitro quanto à ação anti-helmíntica contra Eisenia fetida. O estudo utilizou diferentes concentrações do extrato de AJLE, 25, 50 e 100 mg/mL, o albendazol (10 mg/mL) foi usado como controle positivo e a água destilada serviu como controle negativo. A análise de citotoxicidade do extrato em diferentes doses (µg/mL) contra as linhagens de células do cólon (HCT116) e do fígado (Huh-7) após 48 horas de incubação foi realizada usando um ensaio MTT. O extrato metanólico foi analisado quanto à sua composição fitoquímica usando o equipamento GC-MS. O espectro de GC-MS identificou 19 biomoléculas diferentes. Com relação à atividade anti-helmíntica, a dose mais eficaz de AJLE (100 mg/mL) resultou em paralisia e morte em 7,502±0,812 e 8,190±0,554 minutos, respectivamente. Em comparação, o Mebendazol apresentou menor eficiência, resultando em morte e paralisia em 18,2±0,980 e 13,91±0,373 minutos, respectivamente. A análise histológica dos vermes tratados revelou anomalias significativas na estrutura da superfície. Além disso, o AJLE apresentou efeitos citotóxicos moderados contra as linhas celulares HCT116 e Huh-7, com valores de IC50 de 287,55±2,89μg/ml e 324,2±2,9μg/ml, respectivamente. Esse estudo descobriu que o AJLE é uma fonte rica de ingredientes bioativos e pode ser usado para tratar a infecção por helmintíase.
Palavras-chave:
Artemisia judaica; citotoxicidade; anti-helmíntico; E. fetida
INTRODUCTION
Protozoan and helminthic infections pose significant threats to both mortality rates and economic stability in numerous countries (Radek, 2001). Controlling helminth diseases primarily relies on preventive chemotherapies utilizing drugs such as albendazole, and mebendazole which exhibit efficacy against various protozoa and a wide array of helminth infections. However, their widespread use, these drugs face several challenges, including the development of drug resistance, low bioavailability, and water solubility, significant side effects, and toxicity (Njomo et al., 2010; Hong, 2018). Consequently, there is an urgent need for the development of new drugs to effectively treat helminth infections.
laboratory research often involves the use of epigeic earthworm species, such as E. fetida. It is commonly known as the red wiggler worm and is an excellent model organism for in vitro studies due to its ability to quickly reproduce, short lifespan, low care requirements, and high sensitivity towards various medication (Guideline, 2004).
Plants have been utilized to treat ailments for thousands of years, making them a valuable source of bioactive chemicals. Over 80% of people worldwide utilize herbal for fundamental health care, a trend that has grown over the last three decades decades (Ekor, 2014). A. judaica is an annual small plant that is commonly found in Egypt, Yemen, and Saudi Arabia (Liu et al., 2004; Al-Mustafa and Al-Thunibat, 2008b). It is well-known for its fragrant aroma. Bedouins stand this plant as a healer, and it is frequently used in traditional medicine (Liu et al., 2004, Al-Mustafa and Al-Thunibat, 2008a). According to Qanash et al. (2023) A. judaica is a medicinal plant with antioxidant properties and has traditionally been used as an anti-diabetic drug. Previous studies have shown that A. judaica possesses strong antimicrobial, antiviral, and wound-healing properties (Ahmed et al., 2023; Qanash et al., 2023).
Our current study hypothesis, that Artemisia judaica extracts, due to the biologically active substances it contains, could serve as an anti-helminthic and anti-cancer treatment.
This study aimed to evaluate the phytochemical profiling, cytotoxic, and anthelmintic properties of AJLE on E. fetida in vitro.
MATERIALS AND METHODS
The leaves of A. judaica were collected from Tabuk, Saudi Arabia. The identification of this plant was performed by a botany taxonomist from King Saud University. At room temperature, the plant samples were dried. After that, it was held for extraction, sieved, and processed in an electric mill according to the method of Manikandan et al. (2008). The resulting dried powder (100g) was macerated in 70% methanol for 24 hours at 4ºC, followed by percolation 5-7 times until complete extraction. After filtration, ethanol was separated from the extract using a vacuum evaporator set at 50°C and low pressure. The crude extract was then lyophilized and stored at -20°C until further use.
To analyze the phytochemical constituents of the AJLE, the methanol extract was first dispersed in methanol and subjected to GC-MS studies. Experimental conditions for the GC-MS system have been standardized as follows: TR 5-MS capillary standard non-polar column with dimensions of 30 meters in length, 0.25 mm in internal diameter, and a film thickness of 0.25μm. The flow rate of the mobile phase (carrier gas: He) was set at 1.0mL/min. In the gas chromatography segment, the temperature program (oven temperature) started at 40°C and gradually increased to 250°C at a rate of 5°C/min. The injection volume was 1μl. Samples dissolved in chloroform have been subjected to a mass range of 50-650m/z, and the results have been analyzed using the Wiley Spectral library search program.
A total of 25 earthworms, E. fetida, were gathered from agricultural lands and identified by an expert from the College of Food and Agriculture Sciences at King Saud University. Each Petri dish contained five worms of approximately equal size. Albendazole (10 mg/mL) was used as the positive control, while distilled water served as the negative control. The AJLE extract was prepared in distilled water at concentrations of 100, 50, and 25mg/mL. Paralysis time was recorded when the worms exhibited no movement except when vigorously shaken, and death time was recorded when the worms showed no movement even when vigorously shaken or dipped in warm water (50°C) (Parida et al., 2010).
The small parts of the earthworm body were fixed in 10% buffered neutral formalin and then processed for paraffin embedding, and 4μm thick sections were stained with hematoxylin and eosin (H&E) (Drury and Wallington, 1973). Sections were examined and photographed using a digital camera (DP 73) fitted on an Olympus B×61 microscope (Tokyo, Japan).
HCT116 (colon) cancer and Huh-7 (liver) cells were plated in 96 well plates (104/well) and incubated in a CO2 incubator at 37°C for 24 hours. The cells were then treated with different concentrations of plant extracts including (0, 50, 100, 250, and 500µg/ml). Plant extract or doxorubicin (positive control) was added and the cells were incubated for a further 48 hours. Next, 10μl of MTT (5 mg/ml in PBS) was added to each well. After a further 4h incubation at 37°C, the MTT was removed, and the purple formazan product was dissolved in acidified isopropanol and absorbance was measured at 540nm on a microplate reader (BioTek, USA).
Results were expressed as a percentage of cell viability with respect to untreated control cells (as 100%). The IC50 values were calculated from the dose-response curve using OriginPro software.
One-way analysis of variance (ANOVA) was used to examine the data in SigmaPlot® version 11.0 (Systat Software, Inc., Chicago, IL, USA). Differences between groups were considered significant at a p-value ≤ 0.05.
RESULTS
In the current research, numerous substances were determined by GC-MS analysis of the methanolic extract of A. judaica. Table 1 displays the molecular weight (MW), molecular formula, and retention time (TR) of the active principles. The results revealed the occurrence of 19 distinct biomolecules (Table 1 and Fig. 1), such as: Isophorone, 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-, Piperitone, 4-Hydroxy-3,5,5-trimethylcyclohex-2-enone, 3-(5-Methyl-5-vinyltetrahydrofuran-2-yl)butan-2-ol, 2-Propenoic acid, 3-phenyl-, ethyl ester, Benzeneethanol, 2,5-dimethoxy-, 1,2,3,5-Cyclohexanetetrol, 3-O-Methyl-d-glucose, α-Santonin, Pallensin, 4-Epipallensin, Propanoic acid, 2-methyl-, (dodecahydro-6a-hydroxy-9a-methyl-3-methylene-2,9-dioxoazuleno[4,5-b]furan-6-yl)methyl ester, [3aS-(3aα,6β,6aα,9aβ,9bα)], Furan-2(5H)-one, 4-(4-methyl-1-piperidyl)-5-spiro-cyclohexane-, 6,9,12,15-Docosatetraenoic acid, methyl ester, Diisooctyl phthalate, 1,4-Butanediol, 2,3-bis[(4-hydroxy-3-methoxyphenyl)methyl]-, Pectolinaringenin, 4H-1-Benzopyran-4-one, 5-hydroxy-6,7-dimethoxy-2-(4-methoxyphenyl).
In vitro, the anthelmintic activity resulted in paralysis and death of earthworms due to the loss of movement and sensitivity to external stimuli. The study demonstrated that Artemisia judaica leaf extract (AJLE) exhibited superior anthelmintic efficacy compared to mebendazole when tested against earthworms. Specifically, the most effective dose of AJLE (100mg/mL) resulted in paralysis and death times of 7.502±0.812 and 8.190±0.554 minutes, respectively. In contrast, mebendazole demonstrated lower efficacy with paralysis and death times of 18.2±0.980 and 13.91±0.373 minutes, respectively (Table 2).
GC-MS of the methanolic extract of A. judaica displaying 19 peaks with retention times ranging from 7.08min to 28.67min.
Worms in the control group displayed undamaged and well-organized cuticle and muscle body wall (Fig. 2A). Whereas worms in AJLE (100 mg/ml) treated group showed histological changes represented by disruption of the cuticle and distortion in the form of longitudinal and circular muscles (Fig. 2B). However, when worms were exposed to mebendazole, the cuticle, and muscular body wall were destroyed (Fig. 2C).
Histological changes in the cuticle of E. fetida with various treatments. (A): worms in dist. H2O (control). (B): worms in AJLE (100 mg/ml). (C): worms in Mebendazole. Scale bar = 25μm.
The cytotoxicity of AJLE was studied in vitro versus HCT116 and Huh-7 cell lines at various concentrations (50, 100, 250, and 500 μg/mL) for 48 hours. The IC50 of AJLE was attained at 287.55±2.89μg/ml for the colon (HCT) cell line, while it was 324.2±2.9 μg/ml for the liver (Huh-7) cell line (Table 3). Our research findings suggest that the level of cell viability is positively correlated with the dosage administered. After incubating AJLE with varying dosages with HCT and Huh-7 cell line for 48 hours, the cell viability was significantly lower compared to the untreated cells.
Cytotoxicity (MTT) analysis for tested AJLE at various concentrations (µg/mL) against the Colon (HCT116) cancer and Liver (Huh-7) cell lines after 48 h of incubation
DISCUSSION
Cancer and parasitic infectious diseases pose significant global threats. Researchers worldwide have extensively investigated plants to harness their potential as sources of effective anticancer and anthelminthic agents. Plants possess remarkable chemical diversity and the remarkable ability to synthesize highly intricate novel phytomolecules that often offer significant medicinal or health benefits. (Abdraboh, 2023).
Phytochemical analyses of AJLE were conducted using GC-MS techniques, resulting in the identification of a total of 19 chemical constituents. Our findings indicate that AJLE contains several phytoconstituents belonging to oxygenated monoterpenes and oxygenated sesquiterpenes, including compounds like piperitone and santonin, along with others such as Pallensin and Epipallensin. This is consistent with a study conducted by (Khan et al., 2022).
In the current study, the anthelmintic activity of AJLE was evaluated using earthworms. The cuticle, an essential component of annelids, serves as a protective covering that directly interacts with the environment (Meyer et al., 2021). Mebendazole, as demonstrated by Kern (2003), effectively disrupts the metabolic processes of parasites, crucial for their energy supply. However, recent in vitro research has shown that the anthelmintic efficacy of AJLE at a dosage of 100mg/mL surpasses that of Mebendazole. This superiority is attributed to the presence of active chemicals such as Santonin which is sesquiterpene lactones and piperitone.
Both Santonin and Piperitone exhibit a wide range of biological properties, including insecticidal, repellent, antimicrobial, antibacterial, anti-inflammatory, and antimalarial activities (Hellali et al., 2016; Wang et al., 2019 and Kpadonou et al., 2022). Furthermore, artemisinin and its derivatives, classified as sesquiterpene lactones, have been reported to possess efficacy against various parasitic helminths such as Fasciola spp., Opisthorchis spp., and Clonorchis sinensis (Keiser and Utzinger, 2007; Keiser et al., 2007a, 2007b and Utzinger et al., 2007).
The cuticle and epidermis of earthworms function as crucial barriers between their bodies and the environment, regulating ion passage and controlling the entry of foreign substances (Clauss, 2001). Treatment with AJLE at a concentration of 100mg/mL resulted in cuticle collapse and muscle distortion in earthworms, consistent with findings from a study on A. monosperma, which notably affected the motility, viability, and tegument of E. fetida in vitro (Saleh et al., 2024). This effect may be attributed to sesquiterpene lactones such as artemisinin and Santonin. Artemisinin (ART) and its derivatives have previously demonstrated efficacy against various helminths through mechanisms such as increased worm alkaline phosphatase activity (Spicher et al., 2008), interference with parasite sarcoplasmic/endoplasmic reticulum Ca2+-ATPase PfATP6 activity, disruption of parasite mitochondrial function, modulation of host immune function, and inhibition of angiogenesis (Golenser et al., 2006).
Our research demonstrated that AJLE exhibits anticancer effects against the Huh-7 liver cell line and HCT colon cell line. This effect may be attributed to Santonin, known for its anticancer properties (Arantes et al., 2014). These findings align with those of Nasr, et al. (Nasr, 2020), who found that methanol extracts of A. judaica exhibit anticancer activity against MCF-7 breast, LoVo colon, and HepG2 liver cell lines. Additionally, A. vulgaris has been reported to possess high cytotoxic properties against three cancer cell lines: prostate, colon, and human breast (Nawab et al., 2011).
CONCLUSION
Our findings suggested that AJLE holds promise as a natural remedy as its phytochemical profiling revealed the presence of 19 different biomolecules with anticancer and anthelminthic biological properties. AJLE can be used for helminthiasis infections and potentially other health conditions. Further research is warranted to elucidate the mechanisms of action and optimize the therapeutic potential of AJLE.
ACKNOWLEDGMENTS
This work was supported by the Researchers Supporting Project (RSP2024R3) at King Saud University (Riyadh, Saudi Arabia).
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Publication Dates
-
Publication in this collection
21 Feb 2025 -
Date of issue
Mar-Apr 2025
History
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Received
09 May 2024 -
Accepted
10 Aug 2024
