Ethio. J. Appl. Sci. Technol. Vol(4):65-75(2013) 65

In vitro Nematicidal (Anthelmintic) Property of the Seed
Extracts of Anamirta cocculus (Linn.) Against Pheretima
posthuma (L. Vaill.)
Umer Qadir and Paul, V.I.*
Department of Zoology, Annamalai University, Annamalainagar 608 002, Tamil Nadu, Corresponding author: Dr. V.I. Paul, Associate Professor, Department of Zoology , Annamalai University, Annamalainagar 608 002, Tamil Nadu, INDIA, Tel: +91 9443046081, E-mail: ABSTRACT
Anamirta cocculus is a wild climber having antibacterial and piscicidal properties. It
is used in Ayurvedic and folk medicines. The primary objective of the present study
was to evaluate the in vitro nematicidal or nematodicidal (anthelmintic) activity of
methanol, chloroform and petroleum ether extracts of the seeds of A. cocculus using the
earthworm Pheretima posthuma as an experimental model. After calculating the ½ an hour
LC50s of all the extracts, the time taken for paralysis as well as death of the worms in each
of the extracts was tested using three concentrations each viz.,10, 15 and 20 mg mL–1. All
the three extracts produced significant anthelmintic activities in a dose dependent
manner. The nematicidal activities of the extracts were either significantly higher or
more or less equal to the reference drug Albendazole. Highest nematicidal activity was
shown by the methanol extract (20 mg mL–1) with the paralysis and death time of 30.3
and 60.2 minutes respectively. Moreover, methanol extract exhibited an increased paralytic
as well as nematicidal effect over the reference drug Albendazole at all the given
experimental concentrations. The results indicated the strong probability of the A.
seeds as the potential raw material for developing an effective nematicide that
may be highly useful in controlling the plant parasitic nematodes.
Keywords: Albendazole, Anamirta cocculus, Anthelmint, earthworm, nematicide,
Pheretima posthuma

Umer and Paul 66 historical documents, the plant was used in the treatment of bronchitis, chronic skin Plants and plant products are widely diseases, foul ulcers, dermatophytosis, used in the traditional and folk phthisis, inflammation, vertigo, flatulence, medicines throughout the world. epilepsy, chorea, paralysis of pharynx, leg Similarly, plants form the basis of and respiratory centre, breast cancer and some important drugs that have been mastitis (Krishnaraju et al., 2006; Mati and frequently used in Ayurveda, Siddha and de Boer, 2011; Satya and Paridhvi, 2012). Unani system of medicines. Many of these plants are known to have Due to the easy availability, handling convenience as well as anatomical and and nematicidal or physiological resemblance of earthworms nematodicidal, (antihelmintic) properties. with nematodes, the former have been widely Parasitic nematodes are of serious utilized for evaluating nematicidal (anthelmintic) concern not only to the animal world activity (Sollamann, 1918; Dabadi et al., 2011; but also to many of the economically Ilango et al., 2011; Preeti et al., 2012; Elias et al., important plant species (Ellis et al., 2013). Nematode worms, feed on organic matter, bacteria, insects and plants. Approximately 10% of all nematodes feed Majority of the nematode (helminth) on plants and live around or in the roots. The infections are generally limited to most well known form is the root knot tropical regions and are prevalent nematode (Meloidogyne spp.). This is mainly particularly in third world countries of because of the distinctive galls it causes on tropical regions due to poor helminth infected roots, its wide distribution, and the management practices and can pose wide range of plants that it attacks (e.g., large economic and health problems common vegetables, ornamentals, fruit tree (Dhar et al., 1982; Agrahari et al., 2011). etc.) (Bridge et al., 1990). One of the major Chemical control of nematodes coupled crops suffering from nematode attack is with improved crop management has banana (Musa spp.) and the common plant been the important worm control parasitic nematodes attacking banana are strategy especially for plant parasitic Radopholus Pratylenchus nematodes throughout the world. Helicotylenchus multicinctus and Meloidogyne spp. (Gowen and Queneherve, 1990). Foliar feeder development of resistance in nematodes (Aphelenchoides spp.) is one of the few plant- including helminths (Geert and Dorny, parasitic nematodes which live in and 1995; Coles, 1997) against anthelmintics damage leaves, buds, and other soft tissues of have led to the proposal of screening plant parts that are above the ground phytochemicals for their control. In (Pokharel and Larsen, 2007). Lesion traditional systems of medicine also, nematodes (Pratylenchus neglectus and various indigenous plants and plant Pratylenchus thornei) reduces yield as much parts are used as antihelmintics. as 70% in susceptible spring wheat varieties Anamirta cocculus (Linn.) is a wild with little or no visual symptom climber that has been exploited expression in the crop canopy, whereas variously by human beings for different Pratylenchus vulnus is the most common applications including Ayurvedic and root lesion nematode in fruit crops, folk medicines (Satya and Paridhavi, causing damage in apple, peach, cherry, 2011). It has been suggested to possess and grapes (Mai and Mullin, 1975). anti-inflammatory, anti-fungal, anti-microbial, insecticidal, germicidal, antioxidant and In India nematode attack is prevalent in case of anthelmintic properties (Satya and many crops such as banana, cotton, tomato etc. Paridhavi, 2012). In the While nematode infestation is the basic reason Ethio. J. Appl. Sci. Technol. Vol(4):65-75(2013) 67 for low yield of cotton per hectare in India (319 endosperms were packed in the thimble of kg lint/hectare) as compared to other countries the Soxhlet apparatus and were extracted like Israel (1709 kg lint/hectare) (Gokte- separately with methanol, chloroform and Narkhedkar et al., 2006), it causes root and corn petroleum ether solvents for 72 hrs. Each disease in banana leading to 20-50% fruit yield extract was concentrated using rotary loss (Koshy and Sosamma, 2001; Jonathan and vacuum evaporator and the dried extracts Sreenivasan, 2013). In case of tomato, one of the were stored separately for further bioassays major cause of yield loss in India is root by following Qadir et al. (2014). knot nematode (Saravanapriya and Sivakumar, 2005; Kumar and Khanna, Earthworms
2006). Even though attempts to control plant parasitic nematodes are widespread, biological Adult Indian earthworms, P. posthuma of nearly methods including biopesticides are less equal size (121 cm) having anatomical and common with a few exceptions where physiological resemblance with helminths antagonistic fungi like Arthrobotrys dactyloides (nematodes) (Vigar, 1984; Shivkar and Kumar, have been explored to trap and parasitize plant 2003) were collected from moist soil, Bhopal, pathogenic nematodes (El is et al., 2008). The Madhya Pradesh, India and were brought to widespread method of controlling plant the laboratory and used for the experiment. nematodes is by applying toxic chemicals such as aldicarb, carbofuran, phorate etc., Estimation of LC50
that could end up with environmental as well as health issues (Gokte-Narkhedkar Prior to the commencement of anthelmintic et al., 2006). So there is a need to explore plant activity evaluation, static bioassays were origin biopesticides to curb nematode infestation conducted to calculate ½ an hour (½h) in crop plants. In this context, attempts have median lethal concentration (LC50) of the been made in the present work to evaluate the three solvent extracts of the seeds of A. nematicidal activity of three solvent extracts of cocculus on P. posthuma by following APHA the seeds of the piscicidal plant A. cocculus et al.(1992). Irrespective of their sex, groups by taking the Indian earthworm Pheretima of ten adult P. posthuma were kept in posthuma as an experimental model because sterilized Petri plates in triplicate and static anthelmintic/nematicidal studies are usually bioassays were done with methanol, carried out using P. posthuma as an experimental chloroform and petroleum ether extracts of model (Kirtiman, 2012; Majumder, 2013; A. cocculus seeds. The exposure concentrations Chander et al., 2014; Das et al., 2014). of all the extracts were prepared by dissolving the desired quantity of the MATERIALS AND METHODS
respective extract in mg mL–1proportion in 10% propylene glycol prepared in normal Plant material
saline. Appropriate control groups were also maintained by applying 10% propylene The seeds of A. cocculus belonging to the glycol prepared in normal saline without family Menispermaceae were collected the addition of extracts. The dead worms from the wild from Kerala state, India from each of the Petri plates were removed and were dried properly before the after the completion of the fixed time commencement of the experiment. interval (½h) and the mortality rates were recorded. The ½h LC50 for each of the three Preparation of seed extracts
extracts was calculated using their percentage of mortality by following Probit method of Finney Endosperms were collected from the dried seeds of A. cocculus and were powdered with the help of an electric grinder. Five hundred grams each of the powdered Umer and Paul 68 Anthelmintic bioassay
Statistical analysis
Three different concentrations (10, 15, 20 Chi-square (2) test was used to check the mg mL–1) each of the three extracts were heterogeneity of the data used for calculating the prepared in 10% propylene glycol in normal LC50 values. Results with P<0.05 were saline. Albendazole was prepared by considered as statistical y significant and the 95% dissolving it in normal saline at fiducial level of upper and lower confidence concentrations of 10, 15 and 20 mg mL–1 to limits for each of the lethal concentrations was serve as a reference drug. Experimental also calculated. The obtained data in each group control consisted of 10% propylene glycol in were compared with the respective reference normal saline and normal control consisted group using Student‟s„t‟ test. of normal saline only. The earthworms were washed with normal saline to remove all RESULTS AND DISCUSSION
debris, feacal matter etc., and were acclimatized to the laboratory condition before the Median lethal concentrations of the three experimentation. extracts of A. cocculus seeds against P. posthuma are shown in Table 2. The worms Anthelmintic activity was assayed following were more sensitive towards methanol Nargund (1999) and Nayak et al. (2009) with extract when compared to other extracts some minor modifications using P. and correspondingly it showed the lowest posthuma. Six earthworms each were placed in separate Petri dishes containing three 50, LC90 and LC99 values value. The toxicities of the other two extracts were different concentrations (10, 15, 20 mg mL–1) significantly (P<0.05) lesser than that of each of A. cocculus extracts (methanol, methanol extract. No mortality was observed chloroform and petroleum ether) in in the control groups. triplicate along with standard reference drug (Albendazole), experimental control The summary of the nematicidal activity of and normal control. The worms were the seed extracts (methanol, chloroform and observed for paralysis and death at room petroleum ether) of A. cocculus in terms of temperature. The mean time for paralysis its anthelmintic effect is given in Table 3. It was noted when there was no movement of any kind by the earthworms except when concentrations (10, 15, 20 mg mL–1) of the worms were shaken / prodding vigorously.
methanol, chloroform and petroleum ether Death was confirmed when the worms do extracts of the seeds of A. cocculus showed not even revive in normal saline. The time significant anthelmintic activity. In any case, of death of worms (min) was recorded after the anthelmintic activity was either ascertaining that worms neither moved significantly higher or more or less equal when shaken nor when given external to the reference drug Albendazole (Table stimuli or normal saline that was followed with fading of the body color, which means their cellular death (Preeti et al., 2012). The results from treated groups were compared with reference group, experimental control as well as normal control. Details regarding the composition of the experimental set up are summarized in Table 1. Ethio. J. Appl. Sci. Technol. Vol(4):65-75(2013) 69
Table 1. Composition of the experimental setup (in triplicate)
Six earthworms + methanolic extract (10 mg mL–1) Six earthworms + methanolic extract (15 mg mL–1) Six earthworms + methanolic extract (20 mg mL–1) Six earthworms + chloroform extract (10 mg mL–1) Six earthworms + chloroform extract (15 mg mL–1) Six earthworms + chloroform extract (20 mg mL–1) Six earthworms + petroleum ether extract (10 mg mL–1) Six earthworms + petroleum ether extract (15 mg mL–1) Six earthworms + petroleum ether extract (20 mg mL–1) 10 10 (Experimental control) Six earthworms + 10% propylene glycol in normal saline 11 (Normal control) Six earthworms + normal saline Six earthworms + Albendazole in normal saline (10 mg mL–1) Six earthworms + Albendazole in normal saline (15 mg mL–1) Six earthworms + Albendazole in normal saline (20 mg mL–1) All the extracts produced significant at 20 mg mL–1 with the paralysis and anthelmintic activity in a dose death time of 30.3 and 60.2 min dependent manner. While methanolic respectively. Methanol extract exhibited extract showed minimum time needed an increased paralytic as well as for paralysis and death of the anthelmintic effect over the reference earthworms, petroleum ether extracts drug Albendazole at the given showed maximum time needed for experimental concentrations (Table 3). paralysis and death (Table 3). Methanol The potency of the extract was found to extract at all the three concentrations be inversely proportional to the time (10, 15, 20 mg mL–1) was more effective taken for paralysis and death of worms. than the other two extracts as well as reference drug in their respective concentrations. Highest anthelmintic activity was shown by methanol extract Umer and Paul 70
Table 2
. 1/2h LC50, LC90 and LC99 values of seed extracts of A. cocculus against P.
*Significant at P<0.05. Another important observation was the affect the median lethal concentrations progression in the toughness and rigidity since they lie within 95% confidence limit. of the cuticular layer of the paralyzed The steepness of the slope also indicates a worms that were exposed to the extracts. In sharp increase in the mortality rate with a comparison to the control ones, the small increase in the exposure concentration. cuticular layer of the treated worms Further, on extrapolation of percentage of became tough and rigid as the time mortality, a near 100% mortality of the elapsed. The progression of toughness and worms could be predicted at the rigidity of the cuticular layer intensified as calculated ½h LC90 (Table 2) of the seed the death of the earthworms approached extracts of A. cocculus. Therefore the and could be felt by touching the dead calculated ½h LC99 may be considered as worms. The rate of mortality caused by A. the absolute acute concentration causing cocculus seed extracts points to the relationship 100% mortality in the stipulated exposure between percentage of mortality and time interval. Previous report (Qadir and concentration of the extracts. The Paul, 2014) on controlling the yellow stem significant 2 values (Table 2) indicate that borer of paddy using aqueous extract of mortality rates are not significantly A.cocculus also revealed such toxicological heterogeneous and variables such as characters of A. cocculus seeds. individual variations do not significantly Ethio. J. Appl. Sci. Technol. Vol(4):65-75(2013) 71 Table 3. Summary of the in vitro wormicidal activity (in triplicate) of various extracts of the
seeds of A. cocculus on P. posthuma
Group Concentration Methanol extract Chloroform extract Petroleum ether extract Experimental control (10% propylene glycol in normal saline) Normal control (normal saline) Albendazole (reference) Based on Student‟s „t‟ test; n=18 in each group; comparison made with the respective reference group; NS= not significant; **p<0.01; *p<0.05. In this context, on the basis of the to kill P. posthuma. Irrespective of such results of lethal concentrations assays variations in the potencies, all the three (Table 2) as well as previous reports extracts exhibit significant nematicidal / (Preeti et al., 2012; Majumder, 2013; Das et anthelmintic activities. Methanol extract al., 2014), the treatment concentrations for shows the highest level of nematicidal anthelmintic assays (Table 3) were selected potency as well as paralytic activity and in such a way that it corresponds to the thereby making it the best candidate for lowest concentrations of the commonly formulation of biopesticide for the available reports and at the same time control of nematode infestation in above the ½h LC99. This could ensure plants. Agrahari et al. (2011) also maximum mortality in reasonably observed significant anthelmintic activity minimum time. The three preliminary in the case of methanol extract of Jussiaea extracts of the seeds of A. cocculus hyssopifolia. Recently Qadir et al. (2014) show significantly different durations have observed rich presence of alkaloids, for paralysis and death of the earthworms saponins, phenolic compounds and at different concentrations (Table 3) flavonoids in the methanolic extract of A. indicating significant variations in the cocculus. They have also reported that toxicological potencies of the extracts. while alkaloids are present in the Qadir et al. (2014) also observed the petroleum ether extract, chloroform variation in the toxicities of seven extract possesses saponins and phenolic extracts of A. cocculus seeds and reported compounds. Many of these components that methanol extract was the most toxic are known to have antiseptic and one to inhibit microbial growth. The antimicrobial activities. While alkaloids present study is also in agreement with and phenolic compounds are reported to the reports of Qadir et al. (2014) that have sedative as well as anthelmintic methanol extract is the most potent one Umer and Paul 72 properties, saponins are well known The body surface of nematode is a collagen antimicrobial and antiseptic agents rich extra cellular matrix forming the (Tiwari et al., 2011; Visweswari et al., protective cuticle of the organism and is 2013). Saponins also function as critical for its viability (Page and Winter, molluscicidal as well as anthelmintic 2003). The phenolic compounds are shown agents (Wang et al., 2010; Tiwari et al., to bind with the glycoprotein on the cuticle 2011; Visweswari et al., 2013). The of the parasite (Thompson and Geary, combined action of the various bioactive 1995) or interfere with energy generation compounds present in the methanolic process in helminth parasites by uncoupling extract in the A. cocculus seeds might be oxidative phosphorylation (Martin, 1997) responsible for its prominent nematicidal and cause the death of the parasite. In action than the other two extracts. leather industry, plant origin phenolic According to Puupponen-pimia et al. compounds are commonly used in the (2001) and Doughari (2012); alkaloids tanning operation that imparts structural and phenolic compounds present in alteration and stability to collagen of skin plants have an inherent defensive matrix through its reactivity and hence make the collagen molecule to aggregate into fibers. Such aggregations results in the loss of flexibility in the collagen matrix In fact, the present study indicates that (Vidyadhar et al., 2010). The possibility of the methanol extract of A. cocculus is such a chemical interaction between the more effective than the reference drug nematode cuticle and the phenolic Albendazole in its anthelmintic activity, compounds of A. cocculus extract also making it a potential source of cannot be ruled out. Such an interaction in biopesticide to effectively manage the the nematode parasites could also result nematode parasites of plants. Previous in the aggregation as well as loss of reports indicate that the size of the flexibility in their cuticular collagen organism in many cases is directly leading to its toughness and thereby related to the concentration of the affecting various physiological functions. toxicant needed to kill it and therefore At this juncture, it is important to note the anthelmintic activities are generally toughness experienced in the cuticular layer demonstrated by finding out a of the paralyzed earthworms immediately particular concentration of the extract before the death in the present study. Such that can kill P. posthuma in a given morpho-physiological changes might interval of time based on the concept have contributed to the paralysis and that the same concentration of the death of the exposed worms. extract shall wipe out the tiny helminthes/ nematodes (Bachaya et al., Further, saponins which are present in the 2009; Agrahari et al., 2011; Preeti et al., methanolic and chloroform extracts of A. 2012). Therefore, nematode parasites of cocculus (Qadir et al., 2014) are also plants being comparatively very small reported to have anthelmintic activity. organisms when compared to the According to Bachaya et al. (2009), lethal experimental organism (P. posthuma), a effect of saponins against helminths concentration of the extract that cause (nematodes) may be due to vacuolization mortality in P. posthuma could cause and disintegration of integuments in them. death of the tiny nematode parasites of The minimum nematicidal activity observed in plants if properly exposed to it. This the petroleum ether extract (more or less similar could be attained by treating the to Albendazole) of the present study may be concerned plant/plant part (e.g., attributed to the absence of phenolic rhizome of banana before planting) compounds, saponins and flavonoids and with the extract. presence of alkaloids in it (Qadir et al., Ethio. J. Appl. Sci. Technol. Vol(4):65-75(2013) 73 2014) as the latter affect the central REFERENCES
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Arch Dermatol Res (2005)DOI 10.1007/s00403-005-0584-6 A. Barel Æ M. Calomme Æ A. TimchenkoK. De. Paepe Æ N. Demeester Æ V. RogiersP. Clarys Æ D. Vanden Berghe Effect of oral intake of choline-stabilized orthosilicic acid on skin, nailsand hair in women with photodamaged skin Received: 10 January 2005 / Revised: 20 April 2005Accepted: 23 June 2005 Springer-Verlag 2005