International Journal of Innovative Technology and Exploring Engineering (IJITEE) ISSN: 2278-3075, Volume-9, Issue-2S2, December 2019 677 Published By: Blue Eyes Intelligence Engineering & Sciences Publication Retrieval Number: B11641292S219/2019©BEIESP DOI: 10.35940/ijitee.B1164.1292S219 Abstract— Amputated earthworm, Eudrilus eugeniae, possessing clitellum region has regenerating ability and develop into complete worms. Earthworm regeneration assay was developed based on this phenomenon and utilized to identify the presence of antimitotic compounds from the rhizomes of medicinal plant Acorus calamus. In continuation of that study, rhizomes of A. calamus was extracted with water and crude residue was obtained after evaporation. Subsequently, to separate the compounds from crude residue it was extracted with five different organic solvents individually such as benzene, chloroform, ethyl acetate, ethanol and methanol. All five different fractions were examined on earthworm regeneration assay to identify its antimitotic potential. Fractions extracted with benzene and chloroform did not inhibit regeneration as blastema was observed. In contrast fractions extracted with methanol and ethyl acetate inhibited the development of blastema. It suggests that methanol and ethyl acetate fractions might possess antimitotic compounds. TLC analysis with ethyl acetate fraction revealed two distinct bands. Compounds eluted from TLC plates decreased mitotic index of Allium cepa root tips and supporting the presence of antimitotic compounds. Surprisingly, fractions extracted with ethanol increased the regeneration capacity of the worms. Our results demonstrate that rhizomes of Acorus calamus possess both antimitotic and proliferative compounds. Hence we suggest that earthworms could be used as an in vivo system to screen both proliferative and antimitotic compounds from unknown sources. Keywords- Eudrilus eugeniae, Acorus calamus, earthworm, antimitotic, proliferative, aqueous extract I. INTRODUCTION Medicinal plants are one of the major sources of active ingredients in pharmaceutical products throughout world [1]. Pharmacologically active plant-derived natural products provide leads to the development of new drugs [2]. Angiosperms are known for their scope in providing clinically valuable compounds to treat cancerous growth [3], [4]. Plants have also been source of antimitotic compounds, Revised Manuscript Received on December 15, 2019. *Karuppaiah Palanichelvam, Department of Biotechnology Kalasalingam Academy of Research and Education Krishnan Kovil, India. Email: palanichelvam@klu.ac.in Kamaraj Rajamanikkam, Department of Biotechnology Kalasalingam Academy of Research and Education Krishnan Kovil, India. Email: rajamanikkam94@gmail.com Chandran Rajesh, Department of Biotechnology Kalasalingam Academy of Research and Education Krishnan Kovil, India. Email: rajesh.c@klu.ac.in Ramesh Prakash, Department of Chemistry Kalasalingam Academy of Research and Education Krishnan Kovil, India. Email: pkschem07@gmail.com Narayanan Selvapalam, Department of Chemistry Kalasalingam Academy of Research and Education Krishnan Kovil, India. Email: n.selvapalam@klu.ac.in which have great potential to treat cancerous growth [5]. Screening of anticancerous or antimitotic compounds from natural resources is necessary to find new leads or effective drug molecules [6]. Many plants, cell culture or animal-based methods have been in practice to screen or validate the active compounds from natural products [7], [8], [9]. Cell proliferation assays such as dye exclusion methods, ATP assay, clonogenic cell survival assay and other methods using cell cultures are in use [10]. Well established in vivo systems to study compounds that induce proliferation are rare. Many animals have the ability to regenerate new cells or tissues through cell proliferation [11]. However, earthworms have been recently suggested as one of the simple model systems to study regeneration of new tissues [12]. Earthworm species Eudrilus eugeniae, Eisenia fetida, Lumbricus terrestris and Allolobophora chlorotica have been reported to regenerate brains as well [13]. E. eugeniae is a segmented worm and has good regeneration ability when it possesses clitellum region. We have recently reported that regeneration ability of this worm can be used to validate antimitotic compounds from plant extracts [9]. Single medicinal plant may possess many different pharmacologically significant compounds. Acorus calamus plant is reported to have various compounds and is useful to treat cancer, arthritis, inflammatory diseases, diarrhea, dyspepsia, skin disorders, sinusitis, respiratory problems, fevers, bronchitis and other disorders [14]. Bioactive compounds can be separated either by sequential or differential extraction methods either with organic solvents or water. In this study we have used differential extraction method to separate compounds with antimitotic activity from proliferative activity in A. calamus. Different fractions partitioned from aqueous extract of A. calamus rhizomes have been tested on amputated earthworms. We report here that earthworm regeneration could be used to identify not only compounds with antimitotic property but also with potential in cell proliferation by using the medicinal plant. II. MATERIALS AND METHOD A. Plant materials & Chemicals The rhizomes of A. calamus L. and bulbs of Allium cepa L. were purchased and authenticated as described earlier [9]. All chemicals and solvents were purchased in analytical grade from HiMedia or Merck Ltd., India. Earthworm, an in Vivo System to Screen Proliferative and Antimitotic Compounds Kamaraj Rajamanikkam, Chandran Rajesh, Ramesh Prakash, Narayanan Selvapalam, Karuppaiah Palanichelvam