African Journal of Traditional, Complementary and Alternative Medicines African Ethnomedicines Network ISSN: 0189-6016 Vol. 2, Num. 3, 2005, pp. 326-336

African Journal. Traditional, Complementary and Alternative Medicines Vol. 2, Num. 3, 2005, pp. 326-336  

Research Paper 

THE CONTRIBUTION OF THE ANTICHOLINESTERASE ACTIVITY OF PEDIALANTHUS TITHYMALOIDE TO ITS MOLLUSCICIDAL ACTIVITY

Sudhanshu Tiwari^*, Sunil K.Singh, Ajay Singh

Natural Product Laboratory, Department of Zoology D.D.U.GorakhpurUniversity, Gorakhpur – 273 009 (U.P.), INDIA
Email: sudhansh4@rediffmail.com

Code Number: tc05035

Abstract

Experiments were conducted to evaluate the molluscicidal and anticholinesterase activity of aqueous leaf and stem-bark extract ofPedialanthus tithymaloide (Family- Euphorbiaceae)Lymnaea (Radix) acuminata (Lamarack) and Indoplanorbis exustus (Deshyas) – intermediate hosts of endemic schistosomiasis and fascioliasis diseases in cattle and livestock. The toxic effect of both extracts was time- as well as dose-dependent. There was a significant negative correlation between LC values and exposure periods observed, thus the LC₁₀ values of P. tithymaloide leaf decreased from 24.7 mg/L (24 h) to 15.4 mg/L (96 h); LC₅₀ decreased from 69.4 mg/L (24 h) to 27.4 mg/L (96 h); LC₉₀ decreased from 194.6 mg/L (24 h) to 48.9 mg/L (96 h)  against L. acuminata and LC₁₀ values of P. tithymaloide leafdecreased from 5.5 mg/L (24 h) to 0.6 mg/L (96 h); LC₅₀ decreased from 35.8 mg/L (24 h) to 4.3 mg/L (96 h); LC₉₀ decreased from 233.1 mg/L (24 h) to 32.1 mg/L (96 h) against Indoplanorbis exustus. Similar trend was also observed for the aqueous stem-bark extract of P.tithymaloide. Sub-lethal exposure at 24h and 96h of the aqueous leaf and stem-bark extract of P.  tithymaloide causedsignificant (P < 0.05) time- and dose-dependent inhibition of the activity of enzyme acetylcholinesterase (AChE) in the nervous tissues of L. acuminata after 24 h or 96 h exposure periods.Withdrawal study also shows that there was a significant recovery in the acetylcholinesterase activity in both the tissues of snail after the 7^th day of the withdrawal of treatment.Thus, the aqueous extract of P. tithymaloide leaf and stem-bark are environmentally safe candidate molluscicides for controlling vector snails of schistosomiasis and fascioliasis.  

Key words: Schistosomiasis and Fascioliasis, Pedialanthus tithymaloide,Molluscicidal, Anticholinesterase activity

Many aquatic snails act as vectors for larvae of parasitic treamatodes, which also spend part of their life in water and thereby causing a number of diseases. Two disease carried by aquatic snails, schistosomiasis and fascioliasis cause immense harm to man and his domestic animals (Bali et al., 1986; Singh and Agarwal, 1981).

Fascioliasis, caused by trematode, Fasciola hepatica and Fasciola gigantica,is a very common disease of sheep, cattle, goat and other herbivorus animals throughout the world (Singh and Agarwal, 1981; Tiwari et al., 2004).Schistosomiasis, caused by Schistosoma, is a devastating disease of mankind second only to malaria in its prevalence (Jobin, 1973).Both are transmitted by freshwater intermediate host snails, Lymnaea acuminataand Indoplanorbis exustus(Hyman, 1970).

A sure way to tackle the problem of schistosomiasis and fascioliasis is to destroy the carrier snails and remove an essential link in the life cycle of both trematodes.This can be achieved by using synthetic or plant origin molluscicides (Singh et al., 1996). A number of synthetic pesticides were used for controlling various species of gastropods pest in aquatic environment (Singh et al., 1993a; 1993b). But heavy application of these pesticides has now become problem of the living organism including human beings (Dua et al., 1996; Walliszeuski et al., 1998).Control of snail vector population through plant origin pesticides is a very effective and new tool of Integrated Vector Managment Programme. Botanical molluscicides can be preferred to synthetic pesticides due to their biodegradability,low cost, safe and effective molluscicidal effect (Marston and Hostettmann, 1985).

The plant, Pedialanthus tithymaloide (Family-Euphorbiaceae) locally known as Dudhi in India, is a common medicinal plant of India (Satyavati and Gupta, 1987).

In this study, the molluscicidal activity of the leaf and stem bark ofPedialanthus tithymaloidewas observed against target snails Lymnaea acuminata and Indoplanorbis exustus  and their short and long-term effects on acetylcholinesterase enzyme activity in nervousand hepatopancrease tissues of Lymnaea acuminata.

Materials and Methods

Adult Lymnaea acuminata (2.5±0.5 cm); Indoplanorbis exustus (1.05±0.05 cm) were collected locally from the Ramgarh lake of Gorakhpur district and used as test animal. The collected animals were maintained in glass aquaria containing de-chlorinated tap water for acclimatization to laboratory conditions. The water in aquaria was aerated continuously and changed at every 24 hour. Any dead animal(s) was removed as soon as possible from test container to prevent water fouling.

Pedialanthus tithymaloidewas collected locally from botanical garden of D.D.U. Gorakhpur University Gorakhpur and identified by Prof. S.K. Singh, Department of Botany, D.D.U.GorakhpurUniversity, Gorakhpur, India, where a voucher (voucher number 1031) specimen is deposited.

Preparation of aqueous extract

After collection the leaf and stem bark of P. tithymaloidewas thoroughly washed with water, dried in incubator at 37°C and powdered.The dried powder was stored in airtight desiccators, for further use. The dried powder was extracted in distilled water for one hour, centrifuged at 1000 g for 10 minutes, and only the supernatant was used for treatment.

Toxicity experiment   

Toxicity experiment was performed by using method of Singh and Agarwal (1988); ten snails were kept in glass aquaria containing 3 L de-chlorinated tap water. The snails were exposed for 96 h to four different concentrations of Pedialanthustithymaloide leaf and stem bark. Six aquaria were set up for each concentration. Control animals were reared in similar condition without treatment. Behavioural responses of snails were observed for up to 2 h from the beginning of the treatment. Mortality was recorded at every 24 h, 48 h, 72 h and 96 h. LC₁₀, LC₅₀ and LC₉₀ values, upper and lower confidence limits, slope value, t-ratio, g-factor and heterogeneity were calculated according to probit log analysis method by using POLO computer programme of Russel et al. (1977). Experimental conditions of water was atmospheric temperature 31.0±1.0°C; water temperature 28.5± 0.5°C; pH   7.1 to 7.3; dissolved oxygen7.5 to 8.0 mg/L; free carbon dioxide4.0 to 4.5 mg/L; bicarbonate alkalinity102.0 to 104.0 mg/L (APHA/WEF, 1998).

Treatment protocol for measuring Acetylcholinesterase enzyme activity

Acetylcholinesterase inhibition was measured in the nervous and hepatopancrease tissues of Lymnaea acuminata, after exposure to 27.7 mg/Land 55.5 mg/L (40% and 80% of 24 h LC₅₀) and 11.0 mg/L  and 21.9 mg/L(40% and 80% of 96 h LC₅₀) of Pedialanthus tithymaloide leaf and 13.8 mg/L and 27.6 mg/L(40% and 80% of 24 h LC₅₀) and 2.6 mg/Land 5.2 mg/L(40% and 80% of 96 h LC₅₀) of Pedialanthustithymaloide stem bark. Control animals were kept in similar condition without any treatment. After 24 h or 96 h exposure to 40% and 80% doses of 24 h or 96 h LC₅₀ of P. tithymaloide leaf and stem bark, acetylcholinesterase (AChE) activity was measured in the nervous and hepatopancrease tissues of snail by the method of Ellman et al. (1961), as modified by Singh and Agarwal (1982). Homogenate (50 mg/ml, W/V) was prepared in 0.1 M-phosphate buffer, pH 8.0 for 5 min in an ice bath and centrifuged at 1000 g for 30 min at 4 ⁰C. The enzyme containing supernatant (0.05 ml) was pipetted to a cuvette. To this was added 10 ml (5x10^-4M) of freshly prepared acetylthiocholine iodide solution in distilled water as substrate, 1.45 ml of buffer (pH 8.0) and 0.05 ml of the chromogenic agent, 5:5 dithio-bis-nitrobenzoate (DTNB). The change in optical density at 412 nm, caused by the enzymatic reaction, was monitored for 3 min at 25 ⁰C. Protein estimation was done by the method of Lowry et al. (1951). Enzyme activity has been expressed as µ mol ’SH’ hydrolyzed/min./mg protein. 

Withdrawal experiment

In order to see the effect of 7^th day of withdrawal of treatment, Lymnaea acuminata, were exposed at sub-lethal doses of 55.5 mg/L(80% of the 24 h LC₅₀) for 24 h exposure period and 21.9 mg/L(80% of the 96 h LC₅₀) for 96 hour exposure period of P. tithymaloide leaf and at sub-lethal doses of 27.6 mg/L(80% of the 24 h LC₅₀) for 24 hour exposure period and 5.2 mg/L(80% of the 96 h LC₅₀) for 96 hour exposure period of P. tithymaloide stem bark.

After termination of experiments, 50% of the test animals were sacrificed and the activity of acetylcholinesterase enzyme was measured in nervous and hepatopancrease tissues. The other half was transferred to fresh water free from any treatment, which was changed every 24 h for the next six days. Following this, the activity of acetylcholinesterase enzyme was measured in both nervous and hepatopancrease tissues. Control animals were kept in similar condition without any treatment.

Each experiment was replicated at least six times and data have been expressed as mean± SE. Test of correlation, two-way analysis of variance and Student’s t-test were applied for locating significant differences (Sokal and Rohlf, 1973).

Results

Effect on behavioral Changes

Exposure to leaf and stem bark extracts of Pedialanthus tithymaloidecaused significant behavioural changes in the freshwater snails L. acuminata and I. exustus. Behavioural changes appear within 5 to 15 min of exposure. The initial 30-40 min. was a period of hyperactivity.

Toxicity

Table 1: Toxicity (LC₁₀, LC₅₀ and LC₉₀) of aqueous leaf extract of Pedialanthus tithymaloide against Lymnaea acuminata at different time intervals

After some time they started crawling on each other. As the poison took over, there was a muscular twitching and the snails become spirally twisted. Prior to death, there was complete withdrawal of the body inside the shell. At the highest doses mortality occurred in about 100-150 min. of exposure period. Even with the weaker doses the sequences of events leading to death were the same.

The LC values of aqueous extract of P. tithymaloide leaf and stem bark on snails for periods ranging from 24 hour to 96 h are given in Tables 1- 4.

The toxicity was both time as well as dose dependent. There was a significant negative correlation between LC values and exposure periods. Thus with an increase in exposure time, the LC₅₀ values of leaf extract decreased from 69.4 mg/L (24 h) > 47.2 mg/L (48 h) > 28.1 mg/L (72 h) > to 27.4 mg/L (96 h) in case of L. acuminata (Table 1). LC₅₀ values were also decreased from 35.8 mg/L (24 h) > 23.8 mg/L (48 h) > 13.4 mg/L (72 h) > to 4.3 mg/L (96 h) in case of I. exustus(Table 3).

Table 2: Toxicity (LC₁₀, LC₅₀ and LC₉₀) of aqueous stem bark extract of Pedialanthus tithymaloideagainst Lymnaea acuminata at different time intervals.

Similar trend was also observed in stem bark extract. In this treatment LC₅₀ decreased from 34.5 mg/L (24 h) > 11.1 mg/L (48 h) > 8.6 mg/L (72 h) > to 6.4 mg/L (96 h) against L. acuminata (Table 2). In case of I. exustusLC₅₀ were decreased from 28.2 mg/L (24 h) > 20.5 mg/L (48 h) > 17.2 mg/L (72 h) > to 12.7 mg/L (96 h) (Table 4). 

The slope values given in (Tables 1 - 4) were steep and the separate estimation of LC based on each of the six replicates was found to be within the 95% confidence limits of LC doses. The‘t’ ratio was greater than 1.96 and heterogeneity factor was less than 1.0. The ‘g’ value was less than 0.5 at all the probability levels.

Effect on Acetylcholinesterase activity           

Table 5, shows that treatment of snail L. acuminata with sub lethal doses of aqueous extract of leaf and stem bark of P. tithymaloide for the exposure period of 24 h or 96 h, which caused significant (P<0.05) inhibition in acetylcholinesterase activity in the nervous and hepatopancrease tissues.

Table 3: Toxicity (LC₁₀, LC₅₀ and LC₉₀) of aqueous leaf extract of Pedialanthus tithymaloide against Indoplanorbis exustus at different time intervals.

Thus 24 h exposure to 27.7 mg/L and 55.5 mg/L of P. tithymaloide leaf reduced the AChE activity up to 81 and 70% of controls in nervous tissue; up to 90 and 85% of controls in hepatopancreasetissue of L. acuminata. It was observed that 96 h exposure reduced the AChE activity up to 72 and 56% of controls in nervous;  ; up to 80 and 70% of controls in hepatopancreasetissue of L. acuminata while 24 h exposures to 13.8 mg/L and 27.6 mg/L of P. tithymaloide stem barkinhibited the AChE activity up to 66 and 48% of controls in nervous tissues ; up to  76 and 60% of controls in hepatopancrease tissue of L. acuminata (Table 5). Similarly, 96 h exposureinhibited the AChE activity up to 52 and 34% of controls in nervous tissue; up to 60 and 50% of controls in hepatopancrease tissue of L. acuminata (Table 5).Analysis of variance demonstrated that the inhibition of AChE activity was both time and dose dependent (P < 0.05).

After seven days withdrawal experiment of after exposure to sub-lethal doses of 55.5 mg/Lof leaf and 27.6 mg/L of stem bark for24 h exposure period and for 96 h exposure period at sub-lethal doses of 21.9 mg/Lof leaf and 5.2 mg/L of stem bark (Table 5) shows, there was highly significant (P < 0.05) recovery observed in the acetylcholinesterase enzyme activity in both nervous and hepatopancrease tissues of snail L. acuminata (Figure 1).

Discussion 

The nature and rapid onset of these behavioral responses of snails indicates that both leaf and stem bark extracts perhaps contain some neurotoxins, which might be active at neuromuscular system of the exposed animals. Similar behavioral response was also observed in case of Euphorbia royleana, Euphorbiaantisyphliatica and Jatropha gossypifolia on snail L. acuminata (Singh and Agarwal, 1990; Tiwari et al., 2004). The behavioral changes are indeed reminiscent to the response of snails to organophosphorus and carbamate pesticides (Singh and Agarwal, 1981).

Table 4: Toxicity (LC₁₀, LC₅₀ and LC₉₀) of aqueous stem bark extract of Pedialanthus tithymaloide against Indoplanorbis exustus at different time intervals.

No such behavioral symptoms and death occurred in control groups indicating that no factor other than plant moieties was responsible for altered behavior and mortality in the experimental animals.

Mortality caused by stem bark and latex showed a clear significant positive correlation with dose of the extract. The positive correlation between dose and mortality in leaf and stem bark may be explained by assuming that increase in the concentration of toxicant in aquarium water resulted in more intake or entry of toxicant in the snail body. Stability (life span) of active moiety of pesticides in environment and the rate of their detoxification in animal body also alter the mortality and exposure period relationship (Matsumura, 1985). Increased mortality with increase in exposure periods could be affected by several factors. For example, uptake of active moiety is time dependent, which leads to progressive increase in the uptake of drug and its effect on snail body (Goodmann et al., 1985; Singh and Agarwal, 1988; 1993a; 1993b). Statistical analysis of the data on toxicity brings out several important points. The χ² test for goodness of fit (heterogeneity) demonstrated that the mortality counts were not found to be significantly heterogeneous and other variables, e.g. resistance etc. do not significantly affect the LC values as these were found to lie within 95% confidence limits. The steepness of the slope line indicates that there is a large increase in the mortality of snail with relatively small increase in the concentration of the toxicant. A steep slope is also indicative of rapid absorption and onset of effects. Since the LC values lay within 95% confidence limits, it is obvious that in replicate test of random samples, the concentration response lines would fall in the same range (Rand and Petrocelli, 1988).

Table 5: Inhibition of AChE activity in nervous and hepatopancrease tissues of freshwater snail Lymnaea acuminata exposed to 24 h or 96 h to 40% and 80% LC₅₀ of aqueous extract of Pedialanthus tithymaloide leaf, stem bark, and recovery of AChE activity after 7^th day withdrawal experiment of treatment.

• Values are mean ± SE of six replicates.
• Values in parentheses are % change with control taken as 100%.
• Data were analyzed through student’s test.
• *, Significant (P< 0.05), when treated groups were compared with controls.
• **, Significant (P< 0.05), when withdrawal groups were compared with treated groups.

Animal behaviour is a neurotropically regulated phenomenon, which is mediated by neurotransmitter substances such as AChE (Bullock et al., 1977). The present study shows that aqueous extract of leaf and stem bark of P. tithymaloide caused significant time and dose dependent inhibition in the activity of enzyme acetylcholinesterase in the snails. This enzyme present in synaptic regions and mediates transmission of impulses by breaking acetylcholine into acetic acid and choline (O’Brien, 1976). The acetylcholine at neural and neuromotar regions upon accumulation causes ‘hyper-excitability’ (Kabeer Ahammad Sahib and Ramana Rao, 1980), which in turn might also influence behaviour pattern of animals. Plants belonging to family Euphorbiaceae have significant anti-AChE activity (Tiwari et al., 2004). Kinghorn and Evans (1975) reported that the pharmacological action of Euphorbious plants is due to presence of a group of diterpen phorbal esters, which are reported to promote activity of the enzyme protein kinase C (Aitken, 1987; Evans and Edwards, 1987), which specifically phosphorylates serine and threonine residues in proteins (Aitken, 1987). Since the active site of enzyme contains a serine residue (Koelle, 1975), it is possible that the inhibition of this enzyme is due to the phosphorylation of the active site. So altered behaviors and snail’s mortality in present study is due to the inhibition of enzyme acetylcholinesterase activity in both tissues of the snails.

Thus we can conclude that P. tithymaloideleaf and stem-bark extracts are highly toxic to L. acuminataand I. exustus snails and can be developed as molluscicides for controlling snail’s populations. The reversibility of the action of both extracts despite the high toxicity would be an added advantage in their use.  

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