https://doi.org/10.22319/rmcp.v16i2.6760

Article

Effect of non-aqueous extracts of Artemisia ludoviciana mexicana and Chenopodium vulvaria during experimental murine infection with Hymenolepis diminuta

 

Jesús-Benjamín Ponce-Noguez ^a

Jorge-Luis de-la-Rosa-Arana ^b

Víctor-Johan Acosta Pérez ^c

Geiner-Francisco Álvarez-Sánchez ^a

Froylan Rosales-Martínez ^a

Liliana Alamilla-Beltrán ^d

Benjamín Nogueda Torres ^d

Fabian Ricardo Gómez De Anda ^c*

 

^a Universidad Autónoma de Chiapas. Facultad Maya de Estudios Agropecuarios. Chiapas, México.

^b Universidad Nacional Autónoma de México. Facultad de Estudios Superiores Cuautitlán. Edo. de México, México.

^c Universidad Autónoma del Estado de Hidalgo. Instituto de Ciencias Agropecuarias. Hidalgo, México.

^d Instituto Politécnico Nacional. Escuela Nacional de Ciencias Biológicas. Ciudad de México. México.

 

*Corresponding author: fabian_gomez9891@uaeh.edu.mx

 

Abstract:

The study assessed the effect of non-aqueous extracts of Artemisia ludoviciana mexicana and Chenopodium vulvaria on an experimental infection with Hymenolepis diminuta in mice. Twenty (20) mice were infected with 10 cysticercoids each, which were obtained from Tenebrio molitor fed with feces from infected rats. Egg excretion was monitored for 30 d. Subsequently, the mice were divided into four groups (n= 5): control with DMSO, nitazoxanide (50 mg/kg), Artemisia (375 mg/kg), and Chenopodium (375 mg/kg). Egg shedding began on day 12 postinfection, with a peak between d 19 and 21. After 24 h of treatment (d 30), excretion decreased by 50 %, and at 72 h, by 70 % in the nitazoxanide and Chenopodium groups. At 33 d, the recovery of adult worms was 50 % in the groups with extracts and 25 % in the nitazoxanide group, with damage to the integument and uterine branches. The extracts increased the roughness of the integument without affecting the scolex. The extracts reduced the parasite load, but more studies are required to determine their cestocidal effect or if they only favor the expulsion of the parasite. In addition, it is necessary to evaluate their impact on gastroenteric nematodes. The absence of adverse reactions suggests their potential use in the control of cestodes.

Keywords: Cestode, Anthelmintic, Herbalism, Non-aqueous extract.

 

Received: 07/08/2024

Accepted: 17/02/2025

 

 

Introduction

H. diminuta is a gastroenteric cestode commonly distributed in rats and mice; humans are an accidental host⁽¹⁾. The adult worm settles in the small intestine and has a disarmed scolex, and gravid proglottids are 20 to 60 mm long by 4 mm wide. The round-shaped eggs measure between 60 and 79 μm in diameter and do not have polar filaments. The larval form (cysticercoid) settles in the hemocoel of coprophagous insects, such as fleas (Nosopsyllus and Xenopsylla) and cereal beetles (Tribolium and Tenebrio); however, cockroaches, lepidopterans, myriapods, and coprophilous beetles can also act as intermediate hosts⁽²⁾. Parasitosis is usually asymptomatic; nevertheless, a massive infection can cause catarrhal enteritis. Zoonotic transmission can be prevented by protecting food and water for human consumption to prevent rodent access. Diagnosis is made by coproparasitological identification of eggs⁽³⁾. The feasibility and viability of maintaining and propagating the life cycle of H. diminuta in the laboratory makes it an experimental model with a number of immunological, molecular, and biochemical aspects, among which are the search for alternative treatments to drugs.

In recent years, it has been reported that several of the helminths that impact animals with livestock importance have developed resistance to antiparasitic pharmacological treatment; therefore, several strategies have been implemented to prevent this resistance, ranging from the replacement of the production animal with a breed resistant to parasites to the application of drugs in different rotation and combination schemes⁽⁴⁾. An alternative strategy to control parasitosis is the use of medicinal herbs.

The aromatic herbs Chenopodium spp. (skunk epazote, paico, or Mexican tea) and Artemisia spp. (estafiate) are used in traditional Mexican medicine; the former has been used as an anthelmintic, antispasmodic, and antirheumatic, whereas the latter, in addition to being employed as a diuretic, is used as an analgesic and gastrointestinal anti-inflammatory⁽⁵⁾. The primary constituent of Chenopodium oil is the bicyclic monoterpene ascaridole⁽⁶⁾, which has sedative, antifungal, and analgesic properties with an antiparasitic effect in experimental infections to reduce the parasite load of Cryptosporidium parvum and Eimeria spp. in chickens and H. diminuta in mice⁽⁷⁾. Artemisia essential oils have components with antimicrobial, antioxidant, pesticide, nematicide⁽⁸⁾, and ovicidal activity in mice naturally infected with Hymenolepis nana⁽⁹⁾.

Non-aqueous extracts have been used in the manufacture of cosmetics, nutraceuticals, paints, lubricants, and biodiesel. Vegetable oils, as alternative solvents, are valued for their physicochemical properties, high global availability, biodegradability, low cost, and low toxicity for humans and animals; they are even effective in extracting chlorophyll and antioxidant compounds from avocado leaves for the food industry⁽¹⁰⁾. Therefore, this study aimed to examine the effect of non-aqueous extracts of Artemisia ludoviciana mexicana and Chenopodium vulvaria on experimental murine infection with H. diminuta.

 

 

Material and methods

Hymenolepis diminuta

To propagate the life cycle of H. diminuta, young female rats 6 to 8 wk old weighing approximately 180 to 200 g were experimentally infected. Each rat was given 20 cysticercoids through a gastric tube. The cysticercoids were obtained from the hemothorax of imagoes of Tenebrio molitor. One hundred (100) adult beetles were used because, unlike larvae, adults actively seek food with moisture, thus favoring parasitosis. To obtain the cysticercoids, adult beetles with reddish-brown exoskeletons, recently released from the chrysalis and deprived of food for 12 h, were fed with freshly expelled feces from rats infected with H. diminuta. Ten days later, the insects were sacrificed and the cysticercoids of the hemothorax were recovered; they were counted and administered using the intragastric tube⁽¹¹⁾.

 

Dynamics of expelled eggs

A total of 20 14-mo-old female mice, Mus musculus strain Balb/c, were individually infected gastrically with 10 cysticercoids. To determine oviposition kinetics, the mice’s feces were analyzed for 30 d using a formalin-ether sedimentation concentration coproparasitological technique⁽¹²⁾. Daily, during the early hours of the day, five fecal samples were collected from each mouse and hydrated in physiological saline solution before being processed. To construct the kinetics, it was arbitrarily decided to count the total number of eggs found in three fields at 10x using a brightfield microscope (CME; Leica Microsystems; Wetzlar, Germany) and then the daily average of all samples was calculated.

 

Non-aqueous extracts of Artemisia ludoviciana and Chenopodium vulvaria

Artemisia and Chenopodium extracts were made from the stems and leaves of the dehydrated plant in the flowering stage. The plants were acquired commercially (Las Plantas Medicinales de América; Mexico City, Mexico). The plant matter was pulverized with an Oster® stainless steel blender with a toggle switch (Oster®, model BLST4125, Acuña, Coahuila, Mexico) until particles of 0.2 to 0.7 mm in diameter were obtained. To prepare the non-aqueous extract of each herb, the pulverized biological material was homogenized in corn oil (Maceite, Promotora de Productos y Mercados Mexicanos S.A. de C.V.; Jalisco, Mexico), because it is biodegradable, easily accessible, low-cost, and non-toxic. The extraction was performed using a Thermomix® Kitchen Robot (Thermomix®, model TM31, Vorwerk & Co.; Wuppertal, Germany), programming the following parameters: 60 ºC, homogenizing at 1,100 rpm for 45 min in a ratio of 2 g of dry matter to 5 g of oil, equivalent to 0.4 g/ml. The oil was then filtered using sterile gauze with a vacuum pump to form the non-aqueous extract. Each extract was stored at room temperature in a hermetically sealed amber bottle for 15 d until use⁽¹⁰⁾.

 

Treatment and dosage

On d 30 postinfection, mice infected with H. diminuta were divided into four groups of five individuals each and treated for 3 consecutive days (d 30, 31, and 32 postinfection), determining the number of eggs excreted per animal daily by means of a coproparasitoscopic sedimentation examination as described in the Dynamics of expelled eggs section. Group 1 was administered with 1 % dimethyl sulfoxide (DMSO), group 2 with 50 mg/kg of nitazoxanide (Cryptofin, Laboratorio Aranda, S.A. de C.V., Mexico City, MX), group 3 with the non-aqueous extract of Artemisia, and group 4 with the extract of Chenopodium. Groups 3 and 4 received a dose of 375 mg/kg body weight, according to the dose of highest effectiveness previously reported^(7,13). Animal welfare was conducted in accordance with Mexican regulations for the care and use of laboratory animals⁽¹⁴⁾. At 24 h after the end of treatment administration (d 33 postinfection), adult worms were recovered from each mouse, and the parasite load per host was determined.

 

Evaluation of adult helminths

The recovered helminths were washed with physiological saline solution and fixed for 3 h with 10 % formaldehyde, then placed in a container with 70 % ethanol, where they were kept until their evaluation. The specimens were stained according to the procedure described by Beltrán-Fabián-de-Estrada et al⁽¹⁵⁾, with some modifications. Briefly, each helminth was stained with 1.6 % carminic acid for 15 min, and the excess dye was removed with three washes with 70 % ethanol. Helminths were dehydrated in a “train” of 80, 90, 96 and 100 % ethanol, incubating for 10 min at each step. Finally, the helminths were transported in clove essence for 24 h. The evaluation was carried out in brightfield microscopy to identify morphological alterations in the scolex and in the gravid proglottids of each recovered cestode. In particular, alterations in uterine branches and integuments were sought in proglottids, which was also evaluated by scanning electron microscopy according to a previously described procedure⁽¹⁶⁾. The data obtained were recorded qualitatively.

 

Statistical analysis

All results were expressed as mean values and standard deviation. The analysis of the results between samples was performed with a two-factor ANOVA test; statistical differences were determined at 95 % confidence with the Tukey contrast test (Prism, GraphPad Software, Boston, USA).

 

 

 

Results and discussion

Experimental infection of Tenebrio molitor

Ten days after infection, the beetles were decapitated and a 60 % infection frequency was found. The cysticercoids had an average size of 80 to 100 μm in length. Other authors previously reported an experimental infection frequency of 39 to 41.7 % for Tribolium confusum beetles⁽¹¹⁾; however, as in this study, the dose per beetle was not quantified. The cysticercoids were recovered by manually macerating the hemothorax in saline solution (Figure 1A).

 

 

 

Figure 1: Cysticercoid (A) at 10x and egg (B) at 40x of Hymenolepis diminuta

Panel A shows the pore (A), the tail (B), and the protoscolex (C). Panel B shows the membrane of the hexacanth embryo (A).

 

 

 

Dynamics of expelled eggs

To monitor the establishment of H. diminuta, the fecal samples of each of the experimentally infected mice were analyzed daily for 30 d, prior to the administration of any treatment. The eggs of the cestode were detected from d 12 postinfection and remained present until the end of the experiment. The eggs measured between 30 and 38 μm in diameter (Figure 1B). Other authors have previously reported that the maximum length reached by the helminth is at 9 d postinfection, followed by a decrease in size from d 11 due to the detachment of gravid proglottids⁽¹⁷⁾, which is consistent with the data recorded in this study since egg detection began on d 12 postinfection. Regarding the dynamics of expelled eggs, the data contrast with a 21-d dynamic in experimentally infected rats, where the authors recorded a gradual increase in the number of eggs excreted, starting with 1,106 and reaching 1,722 eggs per gram of feces⁽¹⁸⁾. The kinetics reported here are made up of 30-d data, in which a behavior with increases is observed every 8 d. Figure 2 shows that the largest number of eggs detected was found between days 19 and 21 postinfection.

 

 

Figure 2: Dynamics of Hymenolepis diminuta eggs expelled by experimentally infected mice

 

 

 

Effect of non-aqueous extracts on egg excretion

Overall, a 50 % reduction in egg excretion was observed at 24 h post-treatment; nevertheless, between 48 and 72 h, there was a 75 % reduction in the nitazoxanide and Chenopodium groups, whereas the group treated with Artemisia maintained the 50 % reduction; the analysis of variance of two factors showed statistically significant differences between the groups, so Figure 3 shows the results of the non-significant (ns) and significant comparisons, P<0.05 (*), P<0.001 (***), and P<0.001 (****) of the Tukey contrast test.

 

 

 

Figure 3: Excretion of Hymenolepis diminuta eggs post-treatment

Results obtained with DMSO (D), nitazoxanide (N), Artemisia ludoviciana (A), and Chenopodium vulvaria (C). Non-significant (ns) and significant comparisons, P<0.05 (*), P<0.001 (***), and P<0.001 (****).

 

 

The results with the non-polar extract of Artemisia are similar to those previously published by other authors, where it was observed that in mice experimentally infected with Hymenolepis nana, the aqueous extract of A. absinthium induces, in a dose-dependent manner, the reduction of egg excretion by 61.8 % (400 mg/kg) and 98.8 % (800 mg/kg)⁽¹⁹⁾, whereas the ethanolic extract of A. abrotanum induces a 75 % reduction (150 mg/kg)⁽⁹⁾. Nevertheless, the results of this study contrast with those obtained with naturally infected sheep, where there was a 20 % reduction in nematode egg excretion by using an aqueous extract of A. campestris (1:1.5; 50 ml/animal; 21 d)⁽²⁰⁾. To explain this controversy, at least four factors must be considered: the geographical region of origin of the plant, the anatomical region of the plant from which the extract is prepared, the nature of the extract (aqueous or alcoholic), and the type of parasite under study⁽¹⁹⁾. The increase in antiparasitic resistance in addition to the residual risk of drugs has led to consider that some plants used in traditional medicine could be an alternative to this problem and the first thing that has been sought is to study the plants that have been used in some cultures as an antiparasitic method⁽⁹⁾. Although many plants have been studied, only some have been properly characterized, especially considering that the growing conditions are not necessarily homogeneous worldwide and not necessarily all plant species, even of the same genus, have the same metabolism and therefore the active ingredients may differ in variety and quantity⁽²⁰⁾; they can even vary in the same plant, depending on the anatomical region from which the extract is prepared, and evidently the extraction method, aqueous or ethanolic, will also influence the metabolites obtained.

Once the heterogeneity of the extracts has been considered, it must be borne in mind that the parasites are also heterogeneous and, although the word helminth considers cestodes and nematodes, the former carry out all the nutritive and gaseous exchanges through their integument and its damage will cause irreversible losses in the metabolism. In the case of nematodes⁽²⁰⁾, feeding is carried out by ingestion and their body is surrounded by a very resistant cuticle of scleroprotein composition, which protects the worm from its environment, so the effect of plant extracts is associated with intake.

Regarding the non-aqueous extract of Chenopodium, the results differ from those reported for conventional oil extracts of C. graveolens (100 mg/kg) and C. ambrosioides (400 mg/kg), which showed no reduction in the excretion of H. diminuta eggs after 5 d of treatment⁽⁷⁾. Nonetheless, methanolic and aqueous extracts of C. album induced a gradual reduction (from 86 to 63.5 %) in the excretion of Haemonchus contortus eggs in naturally infected animals treated for 15 d at a dose of 2 mg/kg body weight⁽²¹⁾. Although the viability of the excreted eggs  was  not  proven in  the work  reported here,  it has  been  suggested  that  extracts of C. ambrosioides solubilized with Tween 80 have an ovicidal effect against H. diminuta⁽⁷⁾ and nematicidal effect against H. contortus⁽⁶⁾.

 

Effect of extracts on adult helminth numbers

During the course of infection or treatment, no mice with adverse clinical effects, with pasty or diarrheal feces, were observed, nor was the expulsion of worms observed. The recovered helminths measured between 27 and 35 cm in length (Figure 4B), the size expected in experimentally infected mice⁽¹⁷⁾. In the groups that receive Artemisia and Chenopodium, the number of adults decreased by 50 % (P<0.005), whereas in the nitazoxanide group, it decreased by up to 75 % (P<0.001) compared to the group treated with DMSO. Similar results to those of this work have reported that mice experimentally infected with H. nana and treated with an aqueous extract of A. absinthium obtained a 56.5 % reduction with 400 mg/kg and a 98.5 % reduction with 800 mg/kg⁽¹⁹⁾; in contrast, hydroalcoholic extracts of A. dracunculus and A. absinthium showed in vitro dose-dependent (50 to 250 mg/ml) cestocidal effects against protoscolices of Echinococcus granulosus⁽²²⁾. In this way, Artemisia extract, regardless of the species, can be an alternative strategy for the control of gastrointestinal cestodes; however, the dose at which it should be administered and the characterization of the metabolites that constitute the Artemisia extract are still being studied, which is important because the aqueous extracts of A. herba-alba and C. ambrosioides, the first, administered orally in pigeons, had no effect on the number of Raillietina spp cestodes⁽²³⁾, and the second had no effect on the number of cestodes recovered from naturally infected rodents⁽⁷⁾. This could lead to the proposal of a new strategy for the control of cestode infections through the combination of antiparasitic drugs used in a traditional way and Artemisia extracts, which would also have the advantage of reducing the development of antiparasitic resistance and environmental pollution.

 

 

 

Figure 4: Adult cestodes recovered post-treatment

Results obtained with DMSO (D), nitazoxanide (N), Artemisia ludoviciana (A), and Chenopodium vulvaria (C). In panel A, non-significant (ns) and significant comparisons at P<0.001 (***) and P<0.001 (****). In Panel B, there is a cestode recovered from a mouse treated with DMSO-1%.

 

 

 

Effect on adult helminth integument

Gravid proglottids treated with 1 % DMSO showed a uniform stain that allowed the internal ramifications of uterus with eggs to be observed (Figure 5), whereas scanning electron microscopy showed an integument with uniform striations (Figure 6). Analysis of proglottids treated with nitazoxanide and then stained with carminic acid showed that proglottids retain stain homogeneously, i.e., there are regions with stain clusters and unstained regions, suggesting damage to internal structures. On the other hand, with clove oil, used as a means of lightening tissues, sections of the integument that took on a greenish brown coloration were observed, suggesting damage.

 

 

 

Figure 5: Gravid proglottids of Hymenolepis diminuta post-treatment

The proglottids were stained with carminic acid (left panels) and subjected to scanning electron microscopy (right panels). The figure shows the results obtained with DMSO (D), nitazoxanide (N), Artemisia ludoviciana (A), and Chenopodium vulvaria (C), genital atrium (C) and eggs in the uterine branches (D).

 

 

Figure 6: Scolex recovered 72 hours post-treatment

Results obtained with DMSO (D), nitazoxanide (N), Artemisia ludoviciana (A), and Chenopodium vulvaria (C). Each panel shows the rostellum (A) and suckers (B).

 

 

 

In summary, nitazoxanide caused deformations and abnormal grooves in the proglottid integument, as well as damage to uteruses without eggs. The analysis of the proglottids in scanning electron microscopy showed deformation of the integument associated with fissures and the generation of vesicles that give a velvety appearance without a striation pattern. It has been previously described that lesions in the integument of Taenia crassiceps cysticercoids caused by nitazoxanide treatment consist of separation of the integument from the body of the parasite and the rupture of the vesicle; similarly, scanning electron microscopy shows disintegration of the integument⁽²⁴⁾.

In the proglottids treated with Artemisia, the staining with carminic acid was observed to be uniform; the uterine branches were intact. The integument presented depressions. Scanning electron microscopy showed an increase in integument roughness, areas of fissures, depressions in some portions of the proglottids, small regions with vesicles, and degraded tissue. In 2017, by transmission electron microscopy, Beshay⁽¹⁹⁾ demonstrated that the cestocidal effect (1 mg/ml during 7 h of exposure) of A. absinthium against adult H. nana is associated with morphological changes of the integument due to the accumulation of lipids and, internally, the proglottid showed destruction of the nephridial canal and intrauterine eggs, as well as apoptosis and autophagy processes, which could be attributed to the alteration of nutrient absorption due to integumental damage⁽¹⁹⁾. There are reports of the induction of expulsion of nematodes with macroscopically observable damage when the host is treated with Artemisia⁽²⁵⁾.

In proglottids treated with Chenopodium graveolens, it was observed that carminic acid staining showed unstained areas inside the proglottids, but no damage to the uterine branches or morphological changes in the eggs was observed. With electron microscopy, it was observed that the main change was depressions in the integument; nonetheless, when observing the eggs after staining with carminic acid, they did not show changes in the morphology of the eggs. It has previously been documented that in a dose-dependent manner, Artemisia extract is capable of inducing apoptosis in gravid proglottids of H. nana⁽¹⁹⁾ and thus induce damage in eggs; however, if the damage to the integument is superficial, as in the data reported here, there would be no damage to the eggs. On the other hand, other authors have described that extracts of C. ambrosioides and C. album have a paralyzing effect on the annelids Pheretima posthuma⁽²⁶⁾ and Eisenia foetida⁽²⁷⁾.

At the scolex level, treatment with nitazoxanide showed damage to the suckers and rostellum, as has been reported by other authors using the nitazoxanide-albendazole combination (40 mg/kg) in the murine cysticercosis model. The combination resulted in deformations of the suckers and the rostellum⁽²⁸⁾. The extracts of Chenopodium and Artemisia showed no damage to the rostellum, but the staining with carminic acid showed slight deformation of the suckers. It has been formerly reported by other authors that the extract of Senna alexandrina produces roughness in the proglottids of H. diminuta and deformation in the suckers of the parasite⁽¹⁶⁾.

 

 

 

 

Conclusions and implications

This study determined the effect of non-aqueous extracts of Artemisia ludoviciana mexicana and Chenopodium vulvaria during experimental murine infection with H. diminuta. The extracts reduced the parasite load by 50 % during the first 24 h of administration; nevertheless, with Chenopodium extract, the parasite load is reduced by up to 70 % in the following 72 h. Additional studies should be done to determine doses, active ingredients and, where appropriate, combination with drugs to establish the cestocidal effect. It is also necessary to verify the effect on other genera of gastroenteric cestodes, flukes, and nematodes, including the larval stages. The absence of adverse reactions makes it possible to use the extracts as an alternative strategy for the control of gastroenteric cestode infections.

Acknowledgements

To the Helminthology Laboratory, ENCB, IPN for donating Tenebrio molitor and Hymenolepis diminuta. To the Instrumentation Center of the Center for the Development of Biotic Products, IPN for electron microscopy.

 

 

Conflict of interest

The authors of this paper declare that there is no type of conflict of interest, nor any economic, personal, political, financial, or academic interest that may influence their judgment.

 

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