Print version ISSN 0036-4665
Rev. Inst. Med. trop. S. Paulo vol.54 no.3 São Paulo May/June 2012
Caracterização da atividade moluscicida dos extratos das plantas Bauhinia variegata e Mimusops elengi contra o vetor da Fasciola, Lymnaea acuminata
Kanchan Lata Singh; D.K. Singh; Vinay Kumar Singh
The molluscicidal activity of Bauhinia variegata leaf and Mimusops elengi bark was studied against vector snail Lymnaea acuminata. The toxicity of both plants was time and concentration-dependent. Among organic extracts, ethanol extracts of both plants were more toxic. Toxicity of B. variegata leaf ethanolic extract (96h LC50- 14.4 mg/L) was more pronounced than M. elengi bark ethanolic extract (96h LC50-15.0 mg/L). The 24h LC50 of column purified fraction of B. variegata and M. elengi bark were 20.3 mg/L and 18.3 mg/L, respectively. Saponin and quercetin were characterized and identified as active molluscicidal component. Co-migration of saponin (Rf 0.48) and quercetin (Rf 0.52) with column purified bark of M. elengi and leaf of B. variegata on thin layer chromatography demonstrate same Rf value i.e. 0.48 and 0.52, respectively. The present study clearly indicates the possibility of using M. elengi and/or B. variegata as potent molluscicide.
Keywords: Molluscicidal activity; Plant species; Extracts; Lymnaeid vector; Fasciola; Snail; Lymnaea acuminata; Bauhinia variegata; Mimusops elengi.
A atividade moluscicida das folhas da Bauhinia variegata e da casca do Mimusops elengi foi testada contra o vetor caracol, Limnaea acuminata. A toxicidade de ambas as plantas é dependente do tempo e da concentração. Entre os extratos orgânicos, os extratos de etanol de ambas as plantas foi mais tóxico. A toxicidade do extrato etanólico da folha da B. variegata (96 h LC50 - 14,4 mg/L) foi mais pronunciada do que o extrato etanólico da casca do M. elengi (96h - LC50 - 15,0 mg/L). As frações purificadas em coluna durante 24 h LC50 do B. variegata e da casca do M. elengi foram 20,3 mg/L e 18,3 mg/L, respectivamente. A saponina e a quercentina foram caracterizadas e identificadas como os componentes ativos moluscicidas. A co-migração da saponina (Rf 0,48) e da quercentina (Rf 0,52) com a casca purificada por coluna do M. elengi e as folhas da B. variegata na cromatografia demonstraram o mesmo valor Rf isto é, 0,48 e 0,52 respectivamente. O presente estudo indica claramente a possibilidade de usar M. elengi e/ou B. variegata como moluscicidas potentes.
Fasciolosis is one of the most debilitating zoonotic diseases caused by the liver flukes Fasciola hepatica and Fasciola gigantica14-16,36. Incidence of fasciolosis is very common in the cattle of eastern region of the state of Uttar Pradesh in India24. The fresh water snail Lymnaea acuminata is the intermediate host of the F. gigantica29. This disease is at present emerging or re-emerging in many parts of the world such as Latin America, Europe, Africa and Asia16. Due to more attention on SARS, AIDS, malaria and research in immunological approaches to worm control, a little interest is focused on snail control to minimize the fasciolosis/ schistosomiasis11. One of the possible solutions to control fasciolosis is to disrupt the life cycle of Fasciola by killing the vector snail1,9-11,27,35. The continuous and indiscriminate use of synthetic molluscicides for the control of vector snails has created a long detrimental effect on the aquatic environment22. Therefore, there is a need to develop a safe and eco-friendly counterpart of synthetic molluscicides. Molluscicides of plant origin are now gaining special importance because they are more effective, cheaper and safer to non-target organisms and culturally more acceptable8,27. The present study describes the molluscicidal activity of Bauhinia variegata (Order: Fabales; Family: Fabaceae) and Mimusops elengi (Order: Ericales; Family: Sapotaceae) against vector snail Lymnaea acuminata. Earlier it has been reported that organic (ether, chloroform and ethanol) extracts of B. variegata and M. elengi leaf and bark contain tannin, saponin, glycoside, terpenoids, flavonoids etc.5,6,8,19,25. Although large numbers of pharmacological effects of both the plants have been noted2,13, yet molluscicidal activity of these plants have not been reported till date.
MATERIAL AND METHODS
Plants Used: Fresh leaf of Bauhinia variegata and bark of Mimusops elengi were collected from Gorakhpur (India), washed thoroughly in running tap water and finally with sterile water, shade dried. The dried part of B. variegata leaf and bark of M. elengi were pulverized separately in the electric grinder and the crude powders were obtained, were then sieved with the help of fine mesh cloth. This fine powder was then used separately for toxicity experiments. The specimens were identified and authenticated by department of Botany DDU Gorakhpur University, Gorakhpur, India.
Solvent Extracts: Fifty grams of the leaf of B. variegata and bark of M. elengi were extracted separately with 100 mL of each solvent viz. ethanol (95%), acetone (99%), ether (99.5%) and chloroform (99%) at room temperature for 24 h. Each preparation was filtered separately through sterilized whatman No.1 filter paper9 and the filtered extracts were subsequently evaporated under vacuum. The residues, thus obtained, were used for the determination of molluscicidal activity. The leaf of B. variegata yielded 220 mg chloroform extract, 250 mg acetone extract, 180 mg ether extract, 300 mg ethanol extract M. elengi bark powder yielded 180 mg chloroform extract, 175 mg ether extract, 190 mg acetone extract, 210 mg ethanol extract.
Column purification: One hundred milliliters of B. variegata leaf ethanol extract and M. elengie bark ethanol extract were subjected to silica gel (60-120 mesh, Qualigens glass, Precious Electro Chemindus Private Limited, Mumbai, India) chromatography through 95×45cm column. Seventy five fractions of five milliliters were eluted with ethanol (95%). Ethanol was evaporated under vacuum and the remaining solids obtained were used for the determination of molluscicidal activity of each fraction.
Thin Layer Chromatography: Thin layer chromatography (TLC) was performed by method of SINGH & SINGH31 as modified by JAISWAL & SINGH9 to identify the active component present in B. variegata leaf extract and M. elengi bark extract. TLC was done on 20×20 cm precoated silica gel (Precious Electrochemical Industry. Pvt , Ltd, Mumbai, India). The solvent benzene/ ethyl acetate (9:1 v:v) was used as the mobile phase. Spots of column purified fractions of B. variegata leaf extract and M. elengi bark extract along with their respective active components quercetin and saponin were applied on TLC plates with the help of micropipette. Further, the TLC plates were developed by iodine vapor. Copies of the chromatogram were made by tracing the plates immediately and the retardation factor (Rf) was calculated.
Pure Compound: Quercetin (3,3,4,5,7-penta hydroxyflavone) and saponin (Sapogenin~10-20%) were procured from Sigma Chemical Co. USA.
Collection of test animals: The adult fresh water snails, L. acuminata (2.25 ± 0.20 cm in length) were collected locally from different ponds, lakes and low lying submerged fields in Gorakhpur and were used as test animals. The collected snails were acclimatized for 72 h in the laboratory condition. Experimental animals kept in the glass aquaria containing dechlorinated tap water at 23 ± 1 °C. The pH, dissolved oxygen, free carbon dioxide and bicarbonate alkalinity were 7.1-7.3, 6.5-7.3 mg/L, 5.2-6.3 mg/L and 102-105 mg/L, respectively. Dead animals were removed to avoid any spoilage of the aquaria water.
Concentration-response relationship: The toxicity experiments were performed by the method of SINGH & AGARWAl28. Ten experimental animals were kept in a glass aquarium containing 3l of dechlorinated tap water. Snails were exposed continuously for 96h to different concentrations of B. variegata leaf extract and M. elengi bark extract (Table-1). Six aquaria were set up for each concentration. The control animals were kept in equal volumes of water under similar conditions without treatment. Mortality of snails was recorded at intervals of 24h up to 96h. The mortality of snails was established by the contraction of body within the shell; no response to needle probe was taken as evidence of death. The LC values lower and upper confidence limits (LCL and UCL), slope values, t- ratio, g-values and heterogeneity factor were calculated by using polo computer software of ROBERTSON et al.18 (2007). The regression coefficient between exposure time and different values of LC50 was determined by the method of SOKAL & ROHLF32.
The toxicity of different organic solvent extracts of leaf powder of B. variegata and bark M. elengi was time and concentration dependent. The 24h LC50 of the leaf powder of B. variegata and bark powder of M. elengi was 244.70 mg/L and 91.19 mg/L, respectively (Table-2 & 3). There was a significant (p < 0.05) negative correlation between the LC50 and exposure time. B. variegata leaf ethanol extract (24h LC50- 38.42 mg/L) and M. elengi bark ethanol extract (24h LC50- 44.61 mg/L) were more toxic in comparison to other organic solvents (Table-2 & 3). The column purified fraction of B. variegata and M. elengi were highly toxic. The maximum molluscicidal activity of column purified B. variegata leaf and M. elengi were noted in the 20-30 and 16-26 of the 5 mL Si-gel eluted fractions, respectively. The 96h LC50 of column purified fraction of B. variegata (5.28 mg/L) was higher than the M. elengi (7.20 mg/L) (Table- 2 & 3). The 96 LC50 of quercetin and saponin was 5.28 mg/L and 1.30 mg/L, respectively. Thin layer chromatography analysis demonstrated that the Rf values of quercetin (0.52) was equivalent to the Rf value of column purified fraction of B. variegata (0.52) and saponin (0.48) was equivalent to the Rf values of the column purified fractions of M. elengi (0.48).
The slope values were steep and separate estimates of LC based on each of the six replicates were found to be within 95% confidence limit of LC50. The t-ratio was higher than 1.96 and heterogeneity factor was less than 1.0. The g-value was less than 0.5 at all the probability levels (90, 95, 99). There was significant negative regression (p < 0.05) between exposure time and LC50 of the treatments (Table- 2 & 3).
The present study clearly demonstrates that B. variegata leaf extract and M. elengi bark extract are the potent molluscicides. Mortality caused by all the plant preparations was time- and concentration dependent and there was a negative regression between exposure time and LC values. Toxicity of crude/ purified preparations of both plants against L. acuminata is in the range of potent molluscicide. Thus, high molluscicidal activity, the LC50 being less than 100 ppm8,27. The 96h LC50 of crude preparations of both plants are approximately 100 ppm, whereas all organic extracts LC50 are less than 100 ppm. Among all the organic solvent extracts, the higher toxicity of ethanol extract of B. variegata leaf and M. elengi bark powder indicate that the active molluscicidal component present in the leaf and bark of both plants are more soluble in ethanol than other organic solvents. Molluscicidal activity of B. variegata leaf and M. elengi bark is due to the presence of quercetin and saponin as evident from the individual toxicity and identification by TLC. Earlier, it has been reported that saponins are potent molluscicides20,26,27,33. Pharmacological and biological effects of saponin as antibacterial7, antihelmintic13, antigastric ulcer23 and hypotensive4 have been noted. Methanol extract of Mimusops elengi bark and seed shows significant antifungal activity21. Bark extract of M. elengi showed moderate inhibitory activity against HIV type-1 protease12.
Bauhinia variegata, commonly known as 'cow paw', has great therapeutic properties, mainly due to the presence of flavonoids and other secondary metabolites include terpen, quinines and lactones3. Antiprotozoal, antihelmintic, antitumor, antiulcer and cytotoxic of B. variegata have been reported by CECHINEL FILHO2. Flavones and quercetin have been isolated from leaf of B. variegata17. Quercetin targets cysteine string proteins (CSPα) and impairs synaptic transmission37. The time dependent toxic effect of these plant products may be either due to the uptake of the active moiety which progressively increases the amount of active component in the snails body with increase in exposure period or it might be possible that the active compound could change into more toxic forms in the aquarium water or in the snails body.
A comparison of the molluscicidal activity of quercetin active component present in B. variegata and saponin present in M. elengi with synthetic molluscicides clearly demonstrates that these components are more potent against L. acuminata. The 96 h LC50 of quercetin (5.39 mg/L) and saponin (1.30 mg/L) are lower than those of synthetic molluscicides carbaryl (14.40 mg/L), phorate (15.0 mg/L), formothion (8.56 mg/L) and niclosamide (11.8 mg/L)9,28. 96 h LC50 of saponin (1.30 mg/L) and quercetin (5.39 mg/L) is even lower than active plant molluscicidal components of Allium sativum bulb (271.06 mg/L)31, Cinnamomum tamala (830.90 mg/L)34 Zingiber officinale rhizome (273.80 mg/L), Allium cepa bulb (253.27 mg/L); Trachyspermum ammi (97.59 mg/L)30.
It is evident from the steep slope values that a small increase in the concentration of different treatment causes mortality in snails. A t-ratio value greater than 1.96 indicates that the regression is significant. Values of the heterogeneity factor less than the 1.0 denote that in the replicates lines would fall within 95% confidence limit and thus the model fits the data adequately. The index of significance of potency estimating values indicates that the value of the mean is within the limits at all probability levels (90, 95, 99) as it is less than 0.5.
In conclusion, it can be stated that B. variegata and M. elengi extracts may be used as potent plant molluscicide as their active components are more toxic than their synthetic counterparts. Both plants are found abundantly in this area, so that it is easily available, ecologically safe and culturally more acceptable among native live-stock keepers. Further studies on these plants are needed to verify, whether the extracts of both plants are toxic to other invertebrate (mollusks and aquatic insects) or vertebrate (small fishes) sharing the same habitat with vector Lymnaea. The outcome will certainly give an idea that both plants can be used in aquatic environment with negative ecological consequences. More studies on the mode of action of active molluscicidal components in snail body are also required to explore its full potential as molluscicide.
Authors are thankful to University Grants Commission (UGC), New Delhi, India, for financial assistance (F. No. 39/590-2010 (SR).
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Dr. Vinay Kumar Singh.
Received: 21 January 2012
Accepted: 7 March 2012