ABSTRACT

Fasciolosis is a parasitic disease of world distribution affecting mainly domestic ruminants. The control of this disease is carried out using chemical fasciolicides which, in some cases, have been observed to have environmental problem such us pollution resistance. An alternative is to investigate extracts from plants with anti-Fasciola hepatica effects, taking advantage of the great diversity of flora of our country. The aim of this paper is to identify, assess and elucidate the anti-Fasciola hepatica effect in vitro using antiparasitic extracts of some plants used in Mexican ethno botany. We collected, dried, processed and tested in vitro about 20 plants with their respective chemical elements (hexane, ethyl acetate and methanol), obtaining results of a total of 60 extracts tested. The in vitro evaluations were carried out for 3 days, and the efficacy of each extract was compared with an untreated control group. Each test was repeated 6 times with 13 extracts that showed greater fasciolocide activity. Results from these 13 extracts tested ranged from 80% to 100% activity and the plants tested were: Castela tortuosa (chaparro amargo), Achillea millefolium (plumajillo), Thymus vulgaris (thyme), Justicia spicigera (muicle), Limpia critridora (cedron), Populus alba (Alamo), Mentha piperita (mint), Chenopodium graveolens (epazote de zorrillo), Lippia graveolens (oregano), Artemisia mexicana (estafiate) and Artemisia absinthium (wormwood), which is the hexane variable which showed higher fasciolicide capacity; using a dose of 500 gr/Lt in all the trials. Further in vitro studies should be conducted to obtain the LD50 of each extract to be able to isolate the main active element found in the hexane variable.

1. Introduction

Fasciolosis caused by Fasciola hepatica is a worldwide parasitic disease which hampers animal production and which occasionally infects humans [1]. Its importance lies in the great economic losses ranging from the confiscation of the livers of infected animals to weight loss, growth retardation, reduced production of meat, milk or wool, decreased resistance to other diseases, inhibited reproduction, miscarriages and even death [1-3]. All the above mentioned added to the high costs of deworming makes it one of the costliest parasites of livestock worldwide. Since currently treatment is based on the use of chemical dewormers which often create resistance, poor security and harm to the environment when discarded, alternative methods of control should be studied [4,5]. One alternative is using plants with anthelmintic properties according to ethnobotanical studies [6,7]. Plants have different defensive traits in response to different predators, which tend to be special [8,9]. These mechanisms act with the help of secondary metabolites (they have an unpleasant taste, they inhibit ingestion or assimilation and they are poisonous or sticky) [10,11].

Mexico is in fourth place worldwide in floristic diversity with more than 25,000 recorded species likewise there are over 6000 species of medicinal plants reported so far [12].

According to ethnobotanical studies, plants were selected and crude extracts were prepared from the hexane, ethyl acetate and methanol fragments according to the different polarities from their leaves, stems and flowers [13]. Therefore, based on the fact that in nature wild plants defend themselves against insects, parasites or other organisms by producing toxic substances, it can be stated that the extracts obtained from some Mexican plants used in traditional medicine have an anthelmintic effect of over 80% against the juvenile and adult stages of Fasciola hepatica [14,15].

This study was aimed to identify, assess and elucidate the anti-Fasciola hepatica in vitro with crude extracts of some Mexican plants recorded with antiparasitic activity used in traditional medicine against dysentery, vomiting, vermifuge, nausea, bad absorption, diarrhea and indigestion [16,17].

2. Materials and Methods

2.1. Location of the Study

The plant extract process was carried out in the laboratory of phytochemistry of the UBIPRO in the College the Estudios Superiores Iztacala, and the in vitro evaluations were conducted in the laboratory of Experimental Chemotherapy of the Parasitology Department of the Veterinary Faculty of the National Autonomous University of Mexico.

2.2. Collection of Plant Material

All plants were collected from the states of Puebla and Morelos, (central part of Mexico). The plant material was collected manually by breaking it off, subsequently it was dried in the shade on sheets of paper; then it was in a manual mill and the shredded bark was stored in paper bags.

2.3. Preparation of Extracts

Leaves, flowers and stems were extracted with solvents of different polarity (hexane, ethyl acetate and methanol), using a rota-evaporator. Then three distillations were performed for each fraction every three to four days, depending on the plant. The extracts were concentrated in different vials for later evaluation.

2.4. In Vitro Evaluation

Newly excysted flukes were obtained by artificial excystment of F. hepatica metacercariae. For the evaluation, 5 mg of crude extract of each plant were placed in eppendorf tubes, adding 20 µl of methanol to dissolve the extract and were taken to a concentration of 500 mg/Lt. Culture dishes with 24 wells brand Nunc were used. In each well were 1.6 ml of complete medium (RPMI/bovine serum), 0.2 ml of solubilized extract and 0.2 ml containing 10 flukes. On each plate, 4 control wells were used without treatment containing only the solvent used to dissolve the extracts. In this way the vegetable extracts were tested in quadruplicate and plant extracts that showed activity were reevaluated 7 times to ensure good results. Each trial remained in incubation at 37˚C for 4 days under an atmosphere of 5% of CO₂.

2.5. Interpretation of Test

The flukes were carefully examined on days 1, 2 and 3 using an inverted microscope at 40×. The activity of the extracts was measured by comparing the survival of the treated flukes relative to the untreated control flukes. All procedures were performed under aseptic conditions using a laminar air flow cabinet.

% Efficacy was assessed using the formula (1) below.

When the efficacy rate obtained was above 80%, it was considered that the extract possessed fasciolicide activity.

3. Results and Discussion

(Table 1) Shows the characteristics of the plants used in the in vitro tests, here is the scientific name, common name, the site collects, the parts used to make the extract and its uses in traditional medicine.

It was found, as shown in (Table 2), that from the 60 extracts tested, 11 hexane extract, 1 of methanol and 1 acetate had fascioliscide activity, exerting 80% - 100% mortality between 24 - 72 hours post treatment, is worth mentioning that in all trials was used a control group, which survived up to 7 days.

It is important to note that it was the hexane fraction that exhibited the best capacity; we can therefore assume that it is a metabolite or an active principle that is common in these plants that produce this fascioliscide effect [18].

Finally in (Table 3) shows the plants that had the greatest effect fasciolocide.

These extracts were made from the stems, flowers and leaves of the plants tested avoiding the use of roots which may contain toxic parts of the plant in question [19].

When revising the flukes with the inverted microscope, it was observed that these extracts caused marked relaxation, disintegration of their internal organs and later an imminent disintegration of the body wall. These features

(1)

are also caused by the effect of commercial fascioliscide; it can therefore be assumed that these extracts can cause similar effects [20-22].

With regard to the other fragments (ethyl acetate and methanol) no notorious fascioliscide activity was detected, since very limited activity (10%) was demonstrated during the testing of the extracts.

It is important to point out that all the flukes from the control group remained in perfect condition during the test; hence, it is a fact that these particular medicinal plants had not been tested using in vitro models and that they were used only by a restricted group of people focused on traditional medicine.

Considering the strong evidence of the promising activity exerted under in vitro evaluation, it is recommended that further evaluations be carried out to obtain the LD50 for the determination of the active ingredient(s) which produce the fascioliscide activity [13,23].

4. Conclusion

From 60 plant extracts tested under in vitro conditions at a concentration of 500 mg/Lt against newly excysted Fasciola hepatica metacercariae, 11 from the hexane extracts and 1 ethyl acetate and methanol, showed percentages of 80% to 90% efficacy.

REFERENCES

NOTES

^*Corresponding author.