Background
Helminthiasis is one of the major problem in livestock caused by parasitic diseases viz
., trematodes, cestodes and nematodes. Trematodiasis is often an important problem in small ruminants most probably in the tropical and sub-tropical countries causing heavy mortality and morbidity among livestock. The anthelmintic used against helminth infection is of major concern, since most of them develop resistance against parasites
Haemonchus contortus, Fasciola hepatica and various trematode infection. The anthelminth (Oxyclozanide) a salicylanide compound commonly used as flukicide in veterinary practice developed resistance against amphistomes of sheep due to repeated and improper use of anthelmintic for the worm control programme [
1]. The present study was conducted to compare the efficacy of oxyclozanide and
Marattia fraxinea Sm. against trematode model,
Gastrothylax crumenifer.
Paramphistomosis is a parasitic infection of the domestic and wild ruminants caused by trematodes belonging to the family Paramphistomidae causing economically considerable problems affecting livestock industry by reducing the production [
2]. When immature, the flukes live in the small intestine and abomasum, from where they move to the rumen and become adults. As other digeneans, paramphistomes require snail to complete the life cycle. Snails of the families Planorbidae, Bulinidae and Lymnaeidae acting as intermediate hosts [
3].
The pathogenesis primarily caused by immature stages, which are embedded in the mucosa and attaching mucosa by drawing piece of mucosa in to suckers causing necrosis and haemorrhages [
4]. It causes an acute gastroenteritis and anaemia with high morbidity especially in young animals, particularly small ruminants [
4,
5].
Gastrothylax crumenifer is one of the amphistome, has a world-wide distribution with the highest prevalence in subtropical and tropical areas of Africa, Europe, Russia, Australia and also in South and Southeast Asia [
6]. Chemotherapy is the widely accepted control method however, emergence of resistant strains and increasing concern about drug residues in the food chain have highlighted the need for alternative control strategies. Traditional plant based eco-friendly medicines offer an alternative to chemical based anthelmintics and are reported high percentage of cure with a single therapeutic dose [
7].
. The trials to using plants as anthelmintic remedies go back to older methods of the pre chemotherapeutical periods; however they have become more and more important today [
8]. Several plants have been tested for their anthelmintic efficacies [
9‐
18], against trematode infection. To date, there has been no literature cited to show the anthelmintic property of
Marattia fraxinea Sm., lower vascular cryptogamic plants scientifically investigated to establish whether or not they have anti-trematodal properties in vitro
.
Marattia fraxinea Sm. (family: Marattiaceae) commonly known as tree fern and known for its medicinal properties which is used as a remedy for ankylostomiasis (Nematode Parasite) in Usambara and South Africa [
19]. They are shade loving plants seen under high altitude regions of the world. They have tremendous value in folklore medicine and are widely used as an anthelmintic by the tribal community of Western Ghats region of India.
Most of the parasites have become resistant to chemotherapy, so alternatives are urgently required. Since vaccination has failed in most instances, the search for small molecules is still an option. For malaria and trypanosomiasis quite a number of medicinal plants and isolated natural products have already been tested, but for most of the other parasitic diseases such information is largely missing. Most of the anti-parasitic properties of extracts and isolated natural products have been tested in vitro only. Translation of the in vitro research results into in vivo trials is urgently required. Furthermore, even if animal experiments were successful, we would need clinical trials of the new compounds alone or in combination with established parasiticidal drugs to prove their efficacy and safety. These developments are costly and it is presently difficult to attract the pharmaceutical industries into these fields for various reasons [
20]. The objective of the present work was to investigate the in vitro anthelmintic efficacy of solvent extracts of
M.fraxinea Sm. and subsequent study on morphological changes towards trematode model
Gastrothylax crumenifer.
Discussion
Acetone based extraction showed better phytochemicals from fern. Tannin, Phenol, Terpenoid, Flavonoid, Quinones, steroids, alkaloids and saponins showed strong positivity in acetone fern extract which was the best solvent to express phyto-constituents in this study. Mithraja et al. [
40], performed phytochemical screening with acetone, benzene, chloroform, ethanol, petroleum ether and aqueous extracts of whole plants of
B.orientale, C.thalictroides, D.heterophyllum, D.linearis, H.arifolia, L.ensifolia, N.multiflora, P.calomelanos, P.confusa and leaves and rhizomes of
Drynaria quercifolia, revealed that the presence or absence of the phyto-constituents depend upon the solvent medium used for extraction and the physiological property of individual taxa. The present study on the phytochemical analysis of
Marattia fraxinea Sm. was in confirmation with the earlier study [
40] and stated that tannin containing drugs are used in medicine as astringent and have been found to possess antiviral, antibacterial and anti-parasitic effects for possible therapeutic applications.
Kumudhavalli and Jaykar [
41], evaluated the petroleum ether, chloroform, acetone, ethanol and aqueous extracts of the fern
Hemionitis arifolia (Burm.) Moore, for preliminary phytochemical screening. Our study was subjected for quantifying
Marattia fraxinea Sm. Gracelin et al. [
42], conducted qualitative and quantitative phytochemical analyses in five
Pteris fern species. Qualitative analysis of methanol extract exhibited positivity for 10 phytochemical tests. Present study on the qualitative analysis of ethanolic, petroleum ether acetone, chloroform and aqueous extract of fern species showed acetone extract performed well to exhibit positivity for secondary metabolites. The quantitative analysis of the extract of
Pteris species showed flavonoids content were highest followed by alkaloids and phenolic compounds. The amount of tannin and saponin was very low in the fern extract. However present quantitative study showed terpenoids content was highest and flavonoid content was least in
Marattia fraxinea Sm.
The results of the phytochemical screening and quantitative estimation of the chemical constituents of plant sample have indicated high content of terpenoids, total tannin, total phenol and flavonoids. Terpenes are widespread in nature, mainly in plants as resent constituents of essential oils. Saponin as a group include compounds that are glycosylated steroids, triterpenoids and steroids alkaloids. Many saponin are known to be antimicrobial to inhibit mould, and protect plant from insect attacks. Saponins may be considered as a part of plants defence systems found in plants named phytoanticipins or phytoprotectants [
43]. These structurally diverse compounds have also been observed to kill protozoans and helminths, to be antioxidants and also acts as antifungal and antiviral [
44,
45]. Xanthorrhizol is a bisabolane type sesquiterpenoid compound posses variety of antimicrobial, antioxidant, anthelmintic activity [
46]. Essential oils are formed by aromatic odor as secondary metabolites, composed of terpenes or terpenoids. The cytotoxic activity of essential oils is mostly due to the presence of phenols, aldehydes and alcohols [
47] and are effective against a large variety of organisms including bacteria, fungi, viruses, protozoa as well as metazoan parasites [
48]. Flavonoids are potent secondary metabolites having high anthelmintic activity and the toxicity of most isolated flavonoids in animal cells is very low [
49], several ubiquitous flavonoids genistein, kaemferol, rutin, quercetin etc., showed deleterious effects on selected species of parasitic helminths. Flavonoid kaempferol exerted a strong adulticidal activity on
Schistosoma mansoni [
50]. The anthelmintic activity of flavonoids, genistein, isoflavones found in the root extracts of
Flamingia vestita [
51] mediated its action on cellular/molecular targets in mammals. In flatworms they act on tegumental enzymes causing paralysis and death [
52]. The approved anthelmintics for trematodes are Oxyclozanide, Praziquatel and Triclabendazole, however the rapid spread of triclabendazole resistance is an important motivation for drug discovery of novel trematodicidal drugs [
53].
Dryopteris filix-mas (Dryopteridaceae), contains vermicidal phloroglucinols, such as aspidin, deaspidin and filixic acid are active against intestinal cestodes, paralyze the worm’s tegument [
54]. Pelletierine an anthelmintic alkaloids from
Punica granatum (Lythraceae) and arecoline from
Areca catechu (Arecaceae) target acetylcholine receptors [
20].
Hrckova and Velebny [
55], stated the surface structure of the tegument of trematodes and cestodes represent the potential target sites as the small molecules can be absorbed in the tegument. Athanasiadou and Kyriazakis [
56], stated the secondary metabolites, alkaloids in plants act mainly on adult stage of flukes and showed its antiparasitic effects on some nematode parasites of cattle and goat and also against a digenean fluke of sheep, namely
Paramphistomum cervi. Several studies showed that extracts from plants grown to serve as human food for example:
coconut, onion, garlic, fig, date, annanas, chicory have high anthelmintic potential against intestinal nematodes, cestodes and trematodes [
8].
2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]]- (CAS), PHYTOL, Fern-8-ene, Xanthorrhizol a potent group of terpenoids present in these fern extract under GC-MS analysis might be attributed to significant antioxidant potential and anthelmintic activity. These findings provide quantitative estimation of the phytochemicals as well as mineral element analysis which are important in understanding the pharmacological and/or toxicological actions of medicinal plants [
57].
Various studies on in vitro and in vivo anti-trematodal activity of medicinal plants viz., alcoholic extract of
Allium sativum and
Piper longum [
58],
Balanites aegyptica [
17], bark of
Prosopis cineraria [
18], Plumbagin on
Fasciola gigantica and
Paramphistomum cervi [
37,
59], 50 medicinal plants against
Schistosoma mansoni and
Echinostoma caproni used in Côte d’ Ivoire [
16],
Bombax malabaricum leaves against
Paramphistomum explanatum [
15],
Dregea volubilis leaves against
Paramphistomum explanatum [
60],
Artemisia annua, A.absinthium, A.siminatriloba and
Fumaria officinalis against adult
Schistosoma mansoni, Fasciola hepatica and
Echinostoma caproni in vitro [
14],
Aegle marmelos, Andrographis lineata, A.paniculata, Cocculuc hirsutus, Eclipta prostrata and
Tagetes erecta against
Paramphistomum cervi [
13],
Flemingia estita root tuber against
Paramphistomum sp. [
9]. Similarly in vivo study of five allopathic drug on natural fasciolosis infected cattle [
61], has been reported. However, Pteridophytic plants having anti-trematodal property is reported first time in this study.
Anthelmintics are drugs that cause adverse effects on the helminths which include the effects on vital activities like feeding, neuromuscular transmission, ion exchange or on the tegument [
62]. The normal phytochemicals present in the plant extracts act similar to the mechanisms exhibited by conventional anthelmintics. The phytochemical saponins interact with the cell membranes, causing changes within the cell membranes, and subsequent changes in the cell wall [
63]. Tannins have the capacity to bind to proteins impair vital process like feeding, reproduction of the parasite and disrupt the integrity of the cuticle [
64]. The condensed tannins interact with proline rich proteins on cuticle that will interfere feeding, motility and other key metabolic processes like exsheathment and moulting of the parasites [
65].
The effect of
M. fraxinea Sm. extracts against rumen amphistomes,
G. crumenifer was tested and it was found that complete paralysis and death of the worm occurred at higher concentration (5 mg/ml) with an incubation time of 60 min, compared to positive control taken 10 min incubation time to kill all worms. Gross microscopical changes under stereo-zoom microscope showed test group of fern worms evinced degenerative changes towards suckers, teguments and testes at higher conc (5 mg/ml). Negative control worms showed smooth spineless tegument followed by surface syncytium, subsyncytial zone and longitudinal and circular muscles, as also observed by [
66].
The results of the in vitro incubation study suggests that a dose rate of 1 mg/ml produced negligible changes and 5 mg/ml conc. produced moderate changes, histopathologically in the tegument and affection on muscle integrity when compared to standard drug Oxyclozanide. On microscopic examination, there were effects on the tegument which appeared blebbed, corrugated, bulbous. But the severity of dose rate of 5 mg/ml produced drastic changes in the tegument with blebbing, desquamation, erosion of syncytium and sub-syncytium exposing basal lamina. Histopathological examinations in the present study also suggests affection of the tegument and also the parenchymatous cells, vacoular degenerative changes of Oral sucker which may be depicting an action similar to that of Oxyclozanide where much intense degenerative changes towards testes and ovary noticed. However, the control flukes showed normal microscopic structure of various organs and tegument. The results were in accordance to many other similar works on amphistomes [
17,
35,
66].
The major target organ that was highly affected is the tegument and suckers that damages were observed by LM and SEM. The extract effects were less severe than Oxyclozanide at high concentration (5 mg/ml) The sequences of tegumental surface changes were similar for all doses of extract compared to OXY, that consisted of swelling, fibrous network formation between major and minor folds, blebbing and subsequent rupture, which lead to erosion and desquamation of the tegument, and finally the exposure and disruption of basal lamina. The tegument is an important structure of parasite because it provides covering and protection of the parasite’s body, and supports internal organs. It also controls the secretion, synthesis, perception of sensory stimuli and osmoregulation. It was demonstrated that tegument is a major target of extract, which was probably absorbed by the tegument. The initial tegument swelling was believed to be part of the general response of the fluke to a stress situation, representing an attempt by the fluke to replace damaged surface membrane [
67], caused by osmotic imbalance, due to the disruption of ion pumps present on the apical plasma membrane [
68]. This was followed by swelling, blebbing, disruption, erosion and lesion. Once the surface layer is totally destroyed, the drug could penetrate deeper into the muscular layer and caused motility reduction and cessation that lead to death. Regional difference of responses to the
M. fraxinea Sm. extract were also observed, with the ventral being more severely affected than the dorsal surface, and the anterior and middle third regions as well as the lateral margins of the flukes were generally more suffered than the posterior region. The early changes were found at the oral sucker and the genital pore, which exhibited the swollen appearance and scattered blebs along their rims. The acetabulum also was distorted. Surface changes observed in the present study resembles that demonstrated on
F. gigantica treated with aqueous extract of
Artocarpus lakoocha [
11], and on
P. Microbothrium treated with artemether [
69].
The tegument of
P. cervi comprises an outer surface syncytium underlined by a thick subsyncytial zone and musculature [
70].
M. fraxinea Sm. might be exerted its effect on the tegument first then permeated through the underlying muscle, which exhibited drastically decreased motility. Gross disruption of tegument clearly visible to the naked eye, were observed in all specimens at the higher doses. The fluke’s surface appeared dark and followed by tegumental desquamation. On the other hand, the flukes might also ingest MF through the oral sucker because numerous blebs were frequently found at the anterior part of worm, especially around the oral sucker. Dome-shaped papillae are commonly present on trematodes tegument surfaces, and they are believed to have a sensory function [
71] related to feeding at the oral aperture, pressure detection on the general body surface, and sexual reception around the genital pore [
72]. The papillae were also damaged by
M.fraxinea Sm. which could cause the loss of sensory functions. Besides, the damage of the acetabulum might affect the holding onto the host tissues [
69].