Impact of Taraxerol in combination with extract of Euphorbia tirucalli plant on biological parameters of Lymnaea acuminata

Impacto do Taraxerol em combinação com o extrato da planta de Euphorbia tirucalli nos parâmetros biológicos da Lymnaea acuminata

MOLLUSCICIDES

Impact of Taraxerol in combination with extract of Euphorbia tirucalli plant on biological parameters of Lymnaea acuminata

Impacto do Taraxerol em combinação com o extrato da planta de Euphorbia tirucalli nos parâmetros biológicos da Lymnaea acuminata

Saroj Chauhan; Ajay Singh

Natural Product Laboratory, Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur-273009 (U.P.), India. Telephone: [phone]. E-mail: [e-mail]; [e-mail]

^{Correspondece}

SUMMARY

The present work was carried out to evaluate the molluscicidal activity of active ingredient Taraxerol with the acetone extract of Euphorbia tirucalli against Lymnaea acuminata snail. The (fecundity, hatchability and survivability) of snail L. acuminata exposed to this extract was studied. The effects of the tested extracts on life-history traits of harmful snail L. acuminata have also been evaluated, and this study also expounds the inhibitory effects of these extracts singly as well as in binary combination (1:1 ratio). It concluded that these herbal products act as a potential source of molluscicides, and that they would also have the advantage of easy availability, low cost, biodegradability and greater acceptance amongst users than synthetic pesticide.

Keywords:Lymnaea acuminata; Plant molluscicide; Reproductive activity alteration; Toxicity; Fascioliasis.

RESUMO

O presente trabalho foi levado a efeito para avaliar a atividade moluscicida do ingrediente ativo Taraxerol junto com o extrato de acetona da Euphorbia tirucalli contra o caramujo Lymnaea acuminata. Foram estudados a fecundidade, o desenvolvimento e sobrevivência do caramujo Lymnaea acuminata exposto a estes extratos.

Os efeitos danosos destes extratos testados na vida do caramujo L. acuminata foram também avaliados e este estudo demonstra os efeitos inibidores destes extratos isolados ou em combinação binária (relação l:1). Concluiu-se que estes produtos vegetais agem como fontes potenciais de moluscicidas e que eles também têm a vantagem de obtenção fácil, baixo custo, biodegradabilidade e maior aceitação entre os usuários do que os pesticidas sintéticos.

doi: 10.1590/S0036-46652011000500005

INTRODUCTION

Fascioliasis, a serious infectious parasitic disease infecting domestic ruminants and humans, tops all the zoonotic helminthes worldwide⁹. It is an important disease caused by the liver flukes Fasciola hepatica and Fasciola gigantica, infecting several herbivorous mammalian species, including cattle and sheep etc. Global coupled sourcing of food with changing consumer vogues, including the consumption of raw vegetables and undercooking to retain the natural taste and preserve heat-labile nutrients, has increased the risk of foodborne transmission³⁴. According to the World Health Organization³² the infection was limited in the past to specific and typical geographical areas (endemiotopes), but is now widespread throughout the world.

No doubt that there is now a need to control the veterinary infection along with the human infection⁵. A better way to tackle the problem of fascioliasis is to destroy the carrier snails and remove an essential link in the life cycle of the flukes. Molluscicide application is the most important method of aquatic snail elimination²⁵. Synthetic and natural molluscicides have played a significant role in restricting the population of the snail L. acuminata²⁴. The high cost of imported synthetic compounds, along with increasing concern over the possibility of snail resistance to these compounds and their toxicity in non-target organisms, have given a new impetus to the study of plant molluscicides^6,19. Molluscicides of plant origin have gained greater importance since it is believed that natural products are ecologically and culturally more sound than synthetic ones¹⁹. The use of such local plants as molluscicides will contribute significantly in reducing the burden of purchasing expensive synthetic molluscicides used in snail control²⁰.

The intention of this work is to evaluate the full potential of molluscicidal activity of active compound Taraxerol from plant Codiaeum variegatum and acetone extract of Euphorbia tirucalli singly as well as in binary combination. Study was also done on the reproductive physiology (fecundity, hatchability and survivability) of snail L. acuminata after exposure of the sub-lethal concentrations of these extract.

MATERIALS AND METHOD

Experimental plants: The stem bark of the plant Codiaeum variegatum (Family: Euphorbiaceae) and Euphorbia tirucalli (Family: Euphorbiaceae) was collected locally from Gorakhpur, India. First of all, stem bark was washed with tap water and then dried in the shade. Then the dried stem bark was made into a powder with the help of a mechanical device.

Extraction of Taraxerol from the stem bark of Codiaeum variegatum plant: Dried powder was subjected to extraction through Soxhlet apparatus with petrol solvent for about 70 hours and after 70 hours extraction, the solvent was evaporated by vacuum pump to obtain the extract in solid form. The solid was warmed with CHCl₃ and filtered to remove the CHCl₃ insoluble portion. Then the CHCl₃ soluble portion was concentrated and chromatographed over a silica gel column, and then diluted with petrol, benzene (1:1) to give a crude white solid. The solid was warmed with ether and crystallized from CHCl₃, methanol (1:3 ratios) to obtain Taraxerol (2 Kg powder of stem bark of Codiaeum variegatum yield about 1 gram Taraxerol). The IR and NMR data of Taraxerol was compared with data of Lee et al.¹⁰.

Extraction of compound from Euphorbia tirucalli plant: About 50 grams powder of stem bark was subjected to extraction through Soxhlet apparatus with about 250-300 mL of acetone for about 40 hours at 20-40 °C. When extraction was completed, a small amount of concentrated solution was obtained. This concentrated solution was filtered and the solvent was evaporated at low temperature by using a vacuum pump until the active compound was dried. The dried compound was used for toxicity experiment.

Experimental animal: L. acuminata (2.6 ± 0.3 cm in shell height) were collected from Ramgarh Lake of Gorakhpur district and acclimatized to laboratory conditions for 72h. Thirty experimental animals were kept in glass aquaria, containing dechlorinated tap water for L. acuminata snails. Toxicity experiments were performed using the method of Singh & Agarwal²¹. These snails were exposed continuously from 24h up to 96h to four different concentrations of the extracts. The snails of the control group were kept under similar conditions without any treatment. Mortality was recorded at 24h intervals up to 96h. Lethal concentrations of LC₅₀ values, upper and lower confidence limits (UCL, LCL) and slope values were calculated by computer programme for analysis of bioassay data POLO computer programme of Robertson et al.¹⁷. The regression coefficient was determined between exposure time and different values of LC₅₀.

Toxicity against non-target organism: A mixed population of ten snails (L. acuminata) and ten fish (C. punctatus) were treated in 6L of dechlorinated tap water for 24h with LC₉₀ of Taraxerol and acetone extracts of stem bark of E. tirucalli singly and in binary combination.

Reproductive physiology (fecundity, hatchability and survivability): The fecundity, hatchability and survivability experiment were performed according to the method of Presing¹⁵. In this experiment, L. acuminata snails (2.6 ± 0.3 cm in shell height) were exposed to different sub-lethal concentrations [20% and 40% of LC ₅₀ (24h)] of Taraxerol and acetone extract of E. tirucalli. For the fecundity test glass aquariums were filled with 5L of dechlorinated tap water. Sub-lethal doses of extracts were mixed in each glass aquarium containing dechlorinated tap water. Ten adult snails were placed in each aquarium at room temperature. Six replicates were used for each set of aquarium. Control groups of snails were kept in an aquarium without treatment. Fresh leaf of the plant Nelumbo nucifera (lotus) was floated in each aquarium.

Lymnaeid snails attached ribbon like egg masses (spawns), contain a variable number of eggs ranging from 10-50 to the back surface of the lotus leaf and inner wall of the aquarium when reproducing. The egg masses produced by the snail in the experiment were removed after every 24 hours up to 96 hours and the number of eggs were counted under the microscope. All the spawns of each group were transferred into separate petri dishes (32 cm diameter x 16 cm height) containing one liter of dechlorinated tap water for hatching under the same exposure conditions as above and kept at 25 ± 1 ºC for development of embryo. During the period of embryonic development which lasted from 10 to 13 days, the spawn were examined every day and their growth was studied under a compound microscope in order to follow the progress of development. The survivability of hatched snails was studied for 28 days after hatching.

RESULTS

Toxicological observation

Taraxerol: Lethal concentrations (LC) values of Taraxerol against L. acuminata for periods 24h to 96h are shown in Table 1. Toxicity of Taraxerol was time as well as dose dependent, and there was a significant negative correlation between LC values and exposure periods, LC value decreases as exposure periods increases from 24h to 96h (Table 1).

Acetone extract: LC values of compounds extracted through acetone solvent from plant Euphorbia tirucalli stem bark against L. acuminata for periods 24h to 96h are shown in Table 2. Toxicity of acetone extract of E. tirucalli stem bark was time as well as concentration dependent, and there was a significant negative correlation between LC values and exposure periods, LC value decreases as exposure periods increase from 24h to 96h (Table 2).

Taraxerol+Acetone extracts of stem bark of Euphorbia tirucalli in binary combination (1:1 ratio): Table 3 shows the Lethal concentration values of the effect of combined extracts of Taraxerol+Acetone extracts of stem bark of E. tirucalli (1:1 ratios) against L. acuminata for periods ranging from 24h to 96h, LC value decreases as exposure periods increase from 24h to 96h (Table 3).

Regression coefficient showed that there was a significant (p < 0.05) negative correlation between exposure time and different LC values. There was no mortality in the control group in each set of experiments (Tables 1, 2, 3).

Biological parameters of snails:Treatment of snails with sub-lethal doses (20% and 40% of LC₅₀ of 24h) of Taraxerol, Acetone extract of E. tirucalli stem bark individually and Taraxerol+Acetone extracts of Euphorbia tirucalli stem bark in binary combination (1:1 ratios), fecundity, hatchability and survivability was reduced (Table 4).

DISCUSSION

This study reveals that the active compound Taraxerol when isolated from C. variegatum and acetone extract of E. tirucalli both individually or in combination shows elevated molluscicidal activity. Toxicity data shows that the Taraxerol and acetone extract of E. tirucalli showed a significant positive correlation between concentration and mortality. The concentration dependent response of the snail could be due to several factors, such as rate of penetration, slope, variability and maximal effects of active moieties⁸. Toxicity of these extracts was increased 9.51 times in 24h when mixed in a 1:1 ratio rather than individual treatment of acetone extract of E. tirucalli. Toxicity data also showed a significant positive correlation between the mortality of snail and exposure periods and it is due to several factors, which may be acting separately or conjointly, as uptake of active moiety of plant extracts could be time dependent, which leads to a progressive increase in the titer of the active moiety and its effect in the snail's body^21-23. Stability of active moiety of pesticides in the environment and the rate of their detoxification in an animal body also affect the mortality and exposure period relationship¹².

Statistical analysis of the data on toxicity brings out several important points. The c² test for goodness of fit demonstrated that the mortality counts were not found to be significantly heterogeneous and other variables e.g. resistance etc do not significantly affect the LC₅₀ values, as these were found to lie within the 95% confidence limits. The regression coefficient showed that there was a significant (p < 0.05) negative correlation between exposure time and different LC values. The dose mortality graph exhibits steep slope values. The steepness of the slope line indicates that there is a large increase in the mortality of vectors population with a relatively small increase in the toxicant.

Reproduction is the single most important function in the life cycle of an organism. Successful reproduction determines fitness of organisms. The inability of an organism to complete any one of the reproductive process severely reduces its lifetime reproductive success. Disruption in the reproduction will ultimately affect the abundance and distribution of animals³³. It was also obvious from developmental studies that these plant products do not kill the snails but significantly alter the reproductive physiology of the freshwater harmful snail L. acuminata also, hence it reduces the fecundity, hatchability and survivability of the snail in a dose dependent manner. Reduction in the fecundity was due to intoxication of the parent snail or to the transfer of these plant extracts from the aquatic environment to the egg masses and to the eggs; similar trends in results were also observed by COEURDASSIER et al.⁴. It may also be due to the fact that these plant products probably affected the ovulation hormone in the caudo-dorsal cells in the brain of the snail L. acuminata¹⁸. The lethal concentrations (LC₅₀ and LC₉₀) of nicotinanilide and niclosamide are toxic against different stages of the freshwater snail Lymnaea luteola i. e. eggs, immature and adults²⁶. The reproductive capacity of B. alexandrina is reduced when it was exposed to Allium sativum, A. cepa, and Atriplex halimus^11,30.

The hatchability of eggs was also significantly decreased after exposure to Taraxerol and acetone extract of E. tirucalli than the control group and it may be due to the fact that these plant extracts perhaps induce embryonic malformations, delays in embryogenesis and, for most toxic effects, the death of embryos¹⁶ and hence reduction in hatchability. Sukumaran et al.^27,28,29 also reported the n-butanol extracts of some plant molluscicides like Sapindus trifoliatus, Agave americana, Balanites aegyptica, Jatropha gossypifolia, and Vaccaria pyramidata are as toxic against freshly laid eggs of L. luteola. Mostafa & Tantawy¹⁴ also found a significant reduction in the hatchability of egg masses of snail Biomphalaria alexandrina after exposure to Calendula micrantha and Anagallis arvensis plant and reduction in the hatching of the snails, revealing an interference of latex exposure with the reproductive process of B. glabrata¹³.

Similarly, survivability of hatched snails was also decreased and it may be due to plant products affecting the respiratory function of the snails by acting as uncoupler of oxidative phosphorylation at the mitochondrial level^1,31. The reduction in the growth of treated snails may be due to interference of moluscicides with physiological activities of these snails^2,3,7.

CONCLUSION

It can be concluded that snail control can be made by using these herbal products as potential molluscicides, at sublethal doses these compound also affect the normal reproductive physiology of snails by causing disturbances in their reproduction and development in the life cycle, which ultimately reduces the abundance and distribution of eco-friendly snails.

ACKNOWLEDGEMENTS

One of the authors (Saroj Chauhan) is thankful to the Indian Council of Medical Research, New Delhi (59/24/2006/BMS/TRM), for financial assistance during this work.

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Received: 10 November 2010

Accepted: 22 August 2011

Publication Dates

• Publication in this collection
  13 Oct 2011
• Date of issue
  Oct 2011

History

• Received
  10 Nov 2010
• Accepted
  22 Aug 2011