Gastrothylax crumenifer: Ultrastructure and histopathology study of in vitro trematodicidal action of Marattia fraxinea (Sm.)

Abstract

BackgroundTrematodicidal action of Marattia fraxinea (Sm.) rhizome, extracts against adult Gastrothylax crumenifer and its morphological changes under light and scanning electron microscope was studied in this work. This plant according to folk medicine has been reported to be used as antioxidant, antimicrobial and anthelmintic against ankylostomiasis.MethodsPreliminary screening involved the qualitative methods to detect the presence tannins, saponins, quinones, terpenoids, steroids, flavonoids, phenol, alkaloids, glycosides, cardiac glycosides, coumarins, anthocyanin and betacyanin. Total phenol, total terpenoids, total tannin and total flavonoids were quantitatively estimated. Total phenolic content was estimated by Folin- Ciocalteau method. Total flavonoid content was determined by the aluminium chloride colorimetric method. In-vitro incubation study of Marattia fraxinea Sm. extracts against Gastrothylax crumenifer were performed using 25 ml of Hedon-Fleig (H-F) salt solution containing various concentrations (1, 2, 3, 4 and 5 mg/ml) respectively as test extracts, positive control Oxyclozanide @ 1% (250 mg/25 ml) and negative control H-F salt solution, distributed to 7-Petri plates in an incubator with 5% CO2 at 37 °C. Twenty five amphistomes were incubated and the motility of control and test group was observed under dissection microscope at a regular time interval of (0, 10, 15, 30 and 60) min respectively. The motility response of the parasites was categorized with specific score 3, 2, 1, 0 respectively. Relative Motility (RM) value and Lethal Concentration 50 (LC50) were determined by probit regression analysis. Ultrastructure and histopathological changes on morphology of G.crumenifer were interpreted. The bioactive compounds were analysed using gas chromatography-mass spectrometry (GC-MS) instrument.ResultsPhytochemical analysis of M.fraxinea (Sm.) revealed the presence of secondary metabolites tannins, saponins, quinones, terpenoids, steroids, flavonoids, phenol, alkaloids, cardiac glycosides, coumarins, and betacyanin. Total terpenoid, total phenol, total tannin and total flavonoid content were found to be 16.00 ± 0.85 mg/g. 5.40 ± 0.71) mg GAE/g, 7.90 ± 0.24 mg TAE/g and 1.25 ± 0.41 mg QE/g respectively. GC-MS analysis showed presence of PHYTOL, a diterpenoids, fern-8-ene, a triterpenoids shown to have potent anthelmintic property. In vitro incubation study revealed death of all trematodes, lethal at 60 min incubation time at 5 mg/ml concentration, indicated Relative Motility (RM) value is 0, least effective compared to positive control Oxyclozanide, where RM value is 0 at 10 min. Lethal concentration 50 (LC50) was found to be 4.873 mg/ml. Confirmative study on gross morphology, histopathology, and ultra-structural changes showed considerable effect on suckers, teguments and internal organs and were dose dependent.ConclusionIt is indicative that presence of terpenoids, flavonoids, tannins and phenolics which are contributing factors for anthelmintic activity, producing various degree of damage to suckers, teguments and internal organs. Hence M. fraxinea (Sm.) can be a lead for synthesis of a new trematodicidal drug as alternative for other drugs and overcome anthelmintic resistance and cost effective.