Elsevier

Acta Tropica

Volume 154, February 2016, Pages 19-24
Acta Tropica

Inhibition of acetylcholinesterase and cytochrome oxidase activity in Fasciola gigantica cercaria by phytoconstituents

https://doi.org/10.1016/j.actatropica.2015.10.021Get rights and content

Highlights

Abstract

Fasciolosis is an important cattle and human disease caused by Fasciola hepatica and Fasciola gigantica. One of the possible methods to control this problem is to interrupt the life cycle of Fasciola by killing its larva (redia and cercaria) in host snail. Molecular identification of cercaria larva of F. gigantica was done by comparing the nucleotide sequencing with adult F. gigantica. It was noted that nucleotide sequencing of cercaria larva and adult F. gigantica were 99% same. Every month during the year 2011–2012, in vivo treatment with 60% of 4 h LC50 of phyto cercaricides citral, ferulic acid, umbelliferone, azadirachtin and allicin caused significant inhibition of acetylcholinesterase (AChE) and cytochrome oxidase activity in the treated cercaria larva of F. gigantica. Whereas, activity of both enzymes were not significantly altered in the nervous tissues of vector snail Lymnaea acuminata exposed to same treatments. Maximum reduction in AChE (1.35% of control in month of June) and cytochrome oxidase (3.71% of control in the month of July) activity were noted in the cercaria exposed to 60% of 4 h LC50 of azadirachtin and allicin, respectively.

Introduction

Fasciolosis is important zoonotic disease caused by two major species of fluke Fasciola hepatica and Fasciola gigantica (Agarwal and Singh, 1988, Mas-Coma et al., 2009). Human infection is world-wide (WHO, 2007, Mas-Coma et al., 2009). Human fasciolosis is characterized by hyper eosinophilia, abdominal pain and, exceptionally acute pancreatitis (Saba et al., 2004, Echenique-Elizondo et al., 2005). One way to reduce the incidence of fasciolosis is to de-link the life cycle of fluke, by destroying the Fasciola larvae inside the host body. Human fasciolosis in India was reported in States of Assam, Bihar, Maharashtra, Uttar Pradesh, Arunachal Pradesh and West Bengal (Narain et al., 1997, Elhence et al., 2001, Vatsal et al., 2006, Gandhi et al., 2010, Ramachandran et al., 2012). Singh and Agarwal (1981) reported that 94% of buffaloes slaughtered in Gorakhpur district of U.P., India were infected with F. gigantica. In northern India snail Lymnaea acuminata is the intermediate host of the F. gigantica (Agarwal and Singh, 1988, Sunita and Singh, 2011, Kumar et al., 2012).

Natural products citral, ferulic acid, umbelliferone, azadirachtin and allicin were earlier identified as potent cercaricides (Sunita and Singh, 2011). As a result, the present work is an extension of our previous study in order to elucidate the in vivo effect of these compounds on snail L. acuminata and cercaria larva of F. gigantica.

Control of snail population below a threshold level is one of the major tools to reduce the incidence fasciolosis (Singh et al., 1996a, Mas-Coma et al., 2009, Jaiswal and Singh, 2009). One of the most efficient methods for preventing the spread of fasciolosis is the use of molluscicides (Agarwal and Singh, 1988, Singh et al., 1996a). It is well known that snails are one of the important components of the aquatic ecosystem. Release of molluscicides in aquatic environment against vector snail may also affect the other non target organism. Fasciola larval stages sporocyst, redia and cercaria are the division phase of F. gigantica in snail body. If these larvae will be destroyed by plant molluscicides at sublethal concentration in the snail body, the rate of infection can be reduced without killing the snail. Cercaricidal activity of Zingiber officinale (citral), Ferula asafoetida (ferulic acid and umbelliferone) Allium sativum (allicin) and Azadirachta indica (azadirachtin) against Fasciola larvae was reported by Sunita and Singh (2011) and Sunita et al. (2013). However, mode of the action of these cercaricides inside the cercaria larva is still not known. Our earlier study clearly demonstrated that these cercaricides also act as molluscicidal against snail L. acuminata (Singh and Singh, 1995, Singh et al., 1996b, Singh et al., 1997, Kumar and Singh, 2006). Sublethal treatment with these molluscicides caused significant inhibition of the AChE activity in snail nervous tissue (Singh and Singh, 2000, Kumar et al., 2009). Present study describes the phytotherapy of infected snails by the natural cercaricides and their effect on acetylcholinesterase (AChE) and cytochrome oxides activity in cercaria larva as well as host snail.

Section snippets

Test materials

Acetylthiocholine iodide (ACThI), cytochrome C, DTNB (5,5-dithiobis-(2-nitrobenzoic acid), citral- (3,7-dimethyl-2,6-octadienal), ferulic acid-(4-hydroxy-3-methoxycinnamic), umbelliferone- (7-hydroxycoumarin,7-hydroxy-2H-1-benzopyran-one), azadirachtin (C35H44O16), allicin-3-(allysulfinylthio)-1-proallyl-2-propene-thiosulfinate were purchased from Sigma Chemical Co. USA.

Animals

Adult L. acuminata (2.6 ± 0.20 cm in length) were collected locally. Infected and uninfected snails were separated in two groups.

Acetylcholinesterase in infected snail

In control, snails AChE activity in the nervous tissues of L. acuminata ranged from 0.88 to 0.92 μ mole ‘SH‘ hydrolyzed/min/mg protein (Table 2). In control and treated (60% of 4 h LC50 of citral/ferulic acid/umbelliferone/azadirachtin/allicin) snail L. acuminata no significant change in AChE activity was noted in each month of the year 2011–2012 (Table 2).

Acetylcholinesterase in cercaria larva

In untreated (control) cercaria larva AChE activity ranged from 1.09 to 1.13 μ mole ‘SH’ hydrolyzed/min/mg protein (Table 3). Four hour

Discussion

Nucleotide sequencing of cercaria larva collected from the secondary host L. acuminata and adult F. gigantica from primary host buffalo liver indicate that both cercaria larva and adult fluke found in eastern Uttar Pradesh, India belongs to F. gigantica. There is 99% similarity, in nucleotide sequencing of F. gigantica adult and cercaria larva.

There was no significant change in AChE activity in the nervous tissue of snail L. acuminata exposed to 60% of 4 h LC50 of citral, ferulic acid,

Conclusion

On the basis of above experimental data, it can be conclusively noticed that the larvae of F. gigantica inside the body of vector snail L. acuminata can be killed by citral, ferulic acid, umbelliferone, azadirachtin and allicin. Amongst all azadirachtin and allicin caused maximum inhibition in AChE and cytochrome oxidase in cercaria There is no adverse effect of these active cercaricides on the vector snails, as AChE activity in treated snail's nervous tissues was not significantly altered. As

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