Anthelmintic activity of Cassia auriculata L. extracts-in vitro study

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1 Available online at Scholars Research Library J. Nat. Prod. Plant Resour., 2011, 1 (2): ( ISSN : CODEN (USA): JNPPB7 Anthelmintic activity of Cassia auriculata L. extracts-in vitro study Sucheta A Gaikwad*, Asha A. Kale, Balaso. G. Jadhav, Nirmala. R. Deshpande and Jyoti. P. Salvekar Dr. T. R. Ingle Research Laboratory, Department of Chemistry, S. P. College, Pune ABSTRACT Helminth infections are among the most common infections in humans, affecting a large population of the World. Use of herbal products as antimicrobial agents may provide the best alternative to the wide and injudicious use of synthetic antibiotics. Albendazole, the commercial anthelmintic drug is used as a standard reference and saline as control. As per WHO, only few drugs are frequently used in the treatment of these parasite infections In present study acetone, ethanol, methanol and quath extracts of Cassia auriculata L leaves were investigated for their anthelmintic activity against Ecinia foeitida. Three concentrations (5, 10, 20 mg/ml) of each extract were studied. This experiment involved the determination of time of paralysis and time of death of the worms. The gradual increased in a dose exhibited, a gradual increase in the activity. Quath and ethanol extracts displayed significant anthelmintic activity at highest concentration of 20 mg/ml. It was concluded from the experimental detail study that the plant revealed significant anthelmintic activity, therefore in vivo trials may be conducted for the use in livestock on scientific basis. The data were verified as statistically significant by using two way ANOVA at 1% level of significance (P-value < 0.01) Keywords: C. auriculata L, anthelmintic activity, Ecinia foeitida, leaves extracts. INTRODUCTION Helminthiasis or worm infection is one of the most prevalent diseases in the World. The conditions appear to be worsening with respect to the number of persons affected[1]. The WHO estimates that a staggering two billion people harbor parasitic worm infections. Parasitic worm also infect livestock and crops, affecting food production with a resultant economic impact. Despite this prevalence of parasitic infections, the research on anthelmintic drug is poor[2]. Helminth infections are among the most common infections in humans, affecting a large population of the World Although the majority of infections due to worms are generally limited 62

2 to tropical regions and pose a great threat to health and contribute to the prevalence of malnutrition, anaemia, eosinophilia and pneumonia[3].parasitic diseases cause severe morbidity affecting mainly population in endemic areas with major economic and sol consequences[4]. The gastro-intestinal helminthes becomes resistant to currently available anthelmintic drugs therefore there is a foremost problem in treatment of helminthes diseases[5]. There is an increasing demand towards natural anthelmintics. The plant C.auriculata L. is an aromatic more or less deciduous shrub found through out India up to an altitude of 1,500 m commonly in forests. C. auriculata L. (Caselpinaceae) is a tall, much branched bushy shrub growing wild throughout forests and along roadsides and in wastelands. Literature survey reveals that leaves of C. auriculata L are useful in treatment of leprosy, ulcers and have anthelmintic activity[6]. The anthelmintic activity of quath extract of the leaves is reported[7]. The plant is reported for the presence of polyphenolic compounds.anthelmintic activity, chemically tannins are polyphenolic compounds[8]. Some synthetic phenolic anthelmintics e.g. niclosamide, oxyclozanide and bithionol are shown to interfere with energy generation in helminth parasites by uncoupling oxidative phosphorylation[9]. Another possible anthelmintic effect of tannins is that they can bind to free protein in the gastrointestinal tract of host animal or glycoprotein on the cuticle of the parasite and cause death[10].the present study investigates the anthelmintic activity of the leaves for the different extracts with a view to justify the use of the plant in the treatment of helminths. MATERIALS AND METHODS 1. Plant materials and preparation of extracts Cassia auriculata L was collected from Western Pune, Maharashtra, India. The taxonomic identification was carried out with the help of Flora of Botany Presidency and Flora of Maharashtra (Singh et al.2000) and herbaria were prepared by following standard methods. The specimens were also compared with the authentic herbaria of BSI, Western circle Pune,Maharashtra,India for confirming the identification, its number is BSI/WC/Tech/2009/95. Air shade dried and pulverized plant material was used. Extracts were prepared using exact weighed sample powder in the measured volume of solvents like, acetone, ethanol, methanol and quath.vaccum dried extracs are used for the experiment.solvents used after distillation. 2. Phytochemical screening Qualitative assay, for the presence of plant phytoconstituents such as carbohydrates, alkaloids, glycosides, flavonoids, tannins and saponins were carried out on following standard procedure [7,11]. 3.Collection of Animal The anthelmintic assay was carried as per the method of Ajaiyeoba et. al. [9,14] with minor modifications[12,15]. The assay was performed on adult earthworm (Eicinia fetida) owing to its anatomical and physiological resemblance with the intestinal roundworm parasites of human beings[13,14]. Easy availability of earthworms prompts their extensive use for preliminary in vitro evaluation of anthelmintic compounds[10]. Earthworms were provided and identified by Dept. of Entomology, College of Agriculture, Pune,Maharashtra,India. All earthworms were of approximately equal size (15 cm). They were washed with saline before experiment. 63

3 4. Drugs Albendazole was purchased from G.S.K. Pharma Pvt ltd, Mumbai along with saline NA 0.91 % NaCl. The solvents of analytical grade were used during experimental protocol. Experimental Quath, ethanol, methanol, ethylacetate, extracts of Plant material investigated for their anthelmintic activity against Ecinia foeitida. The anthelmintic assay was carried as per the method of Ajaiyeoba et. al.[9] with minor modifications. All the test solutions and standard drug solutions were prepared freshly before starting the experiment. Six groups of earthworms of approximately equal size were released in to 25 ml solutions of three different concentrations (5,10,20 mg/ml) in petri dishes containing above solutions of extracts. Albendazole was used as reference standard and saline as control. Determination of time of paralysis and time of death of the worm were done. Time for paralysis was noted when no movement of any sort could be observed except when the worms were shaken vigorously. Time for death of worms was recorded after ascertaining that worms neither moved when shaken vigorously nor when dipped in warm water (50 o C) followed with fading away of their body colours. Ethanopharmocological Relevence The plant species screened in this research was recorded by several indigenous medicinal practitioners as antiparasitic, anticancer and/or related activities to the human major organ system. Table I Shows Anthelmintic Activity results of Cassia auriculata L. Extracts. Eicinia foeitida (Earthworms) Extracts Concentrations mg/ml Time for paralysis(p) in min.(mean &SEM) Time for death (D)in min. (Mean &SEM) Control(Saline) ± ±0.022 Quath 10 61± ± ± ± ± ±0.022 Ethanol 10 45± ± ± ± ± ±0.020 Methanol 10 85± ± ± ± ± ±0.013 Acetone 10 42± ± ± ± ± ±0.021 Std. Albendazole ± ± ±0.025 Values are mean ± S.E.M. from six observations. P: Time for Paralysis (min), D: Time for Death of worms (min) The data were verified as statistically significant by using two way ANOVA(TableII) at 1% level of significance (P-value < 0.01) 64

4 RESULTS AND DISCUSSION In conclusion, this study suggests that the plants used by tribals traditionally to treat intestinal worm infections, showed significant anthelmintic activity. The experimental evidence obtained in the laboratory model could provide a rationale for the traditional use of these plants as anthelmintic. The extracts of leaves displays a significant anthelmintic activity in dose dependent manner ( TableI). The anthelmintic activity of ethanolic and quath extracts was comparable with that of standard drug. Albendazole, this drug is effective in a broad range of helminth infections, including round worms, hookworms, whipworms, pinworms and animal side effects. The drug is poorly absorbed and efficacy depends on transit time in the gastrointestinal tract. The toxicity is extremely low, but the drug has not been studied in children under two years of age. Gastro intestinal disturbances may occur during expulsion of the worms, the mechanism of action involves inhibition of the glucose uptake system leading to a lethal depletion of energy reserves in the helminthes. Phytochemical screening of the crude extracts reveales the presence of flavonoids and polyphenolic compound as one of the major chemical constituents. It is possible that tannins contained in the extracts of Cassia auriculata L produced similar effects. Graph I Shows Comparative study on anthelmintic activity of Cassia auriculata L. Extracts on adult Indian earthworm. Time (Min) Treatments Table II Shows Statistical varience of analysis (ANOVA) on anthelmintic activity of Cassia auriculata L. Extracts Source of Variation SS df MS F P-value F crit Between groups Within groups Total Each value represents mean ±SEM(N=6).Data are found to be significant by testing through two way ANOVA with replication at 1% level of significance. 65

5 CONCLUSION It is concluded based on the findings of the present study that the leaves of Cassia auriculata L. possess varying degree of anthelmintic activities. The dose of the extract is increased, a gradual increase in the activity of extracts is dose dependent.(graphi) Quath and ethanol extracts exhibited significant anthelmintic activity at C.auriculata highest concentration of 20 mg/ml. This study strongly supports the traditional use of leaves as anthelmintic. Acknowledgement The authors are thankful to the Principal, S.P.College and Head, Department of Chemistry, S.P.College, Pune-30 for providing experimentation facilities. REFERENCES [1] William o Foye, Principles of Medicinal Chemistry, Varghese Publishing House, Bombay, Third Edition year pg [2] Aswar Manoj, Aswar Urmila,Watkar Bhagyashri.,et al. Int J Green Pharm, ,( 2008) [3]Bundy D. A., Trans Royal Soc Trop Med Hyg, 1994, 8, [4]Tagbota S, Townson S., Adv Parasitol, 2001, 50, [5]Sondhi, S.M., Shahu, R., Magan Archana, Indian Drugs, 1994, 31(7), [6]Nadkarni A.K., Indian Material Medica, Popular Prakashan Pvt. Ltd. Bombay., 1954, Vol. I, 168. [7]Satish B. Kosalge, Ravindra A. Fursule, International Journal of Pharm Tech Research Vol.1,No.1,pp 68-72, Jan March 2009 [8]Dash G. K., Suresh P, Kar D. M., Ganpaty S., Panda S. B., J Nat Rem, 2002, 2, [9]Bate-Smith EC., J Soc Bot, 58: ,(1962). [10]Martin RJ., Vet J, 154:11-34, 1997 [11]Mali R.G., Wadekar R.R., Indian J Pharm Sci. 2008, Jan-Feb, [12].Kokate CK.Practical Pharmacognosy, 4 th edn, Vallabha Prakashan, New Delhi, , (1999). [13]Khandelwal KR. Practical Pharmacognosy Technique and Experiments, 2nd edn, Nirali Prakashan, Pune, , (2000). [14] Ajaiyeoba EO, Onocha PA, OlarenwajuOT., Pharm Biol, 39: ,(2001). [15] Deore SL, Khadabadi SS, Kamdi KS. et al. Int J Chem Tech Res, 1: ,(2009).. 66