We report here that earthworm can be used as an in vivo system to prescreen antimitotic compounds. The known antimitotic compound colchicine and the aqueous extract of Acorus calamus L. rhizome were used to examine this system. The antimitotic activity of colchicine and the rhizome extract was confirmed by methods with Allium cepa root tip, earthworm regeneration, and MTT (3-(4,5-dimethyl thiazolyl-2-yl)-2,5-diphenyltetrazolium bromide). An earthworm regeneration assay examined the regeneration ability of tissues from amputated regions of the earthworm. All 3 assays showed that cell division is inhibited with colchicine and the aqueous extract of A. calamus rhizome. Histology studies with amputated earthworms confirmed that the development of the blastema was observed only in control worms. Immunohistochemistry analysis with proliferation cell nuclear antigen and phospho serine 10 histone H3 antibodies showed that amputated worms treated with colchicine or the aqueous extract of A. calamus did not have active cell division. MTT assay with the MCF-7 cell line (human breast carcinoma) further confirmed that the aqueous extract of rhizome affected cell proliferation. Our results suggest that the earthworm could be used to prescreen the antimitotic potential of plant extracts or other unknown compounds. It is simple, reproducible, and cost-efficient compared to animal cell line-based methods.
To identify antimitotic compounds from abundant and inexpensive plant resources, banana pseudo-stem was (BPS) chosen. Onion root tip assay and earthworm regeneration assay were carried out to test theantimitotic potential of aqueous extract of BPS.Earthworm (Eudrilus eugeniae) regeneration assay exploits the regeneration ability of amputated earthworms that retain the clitellum region. Aqueous extract of BPS decreased the mitotic index in Allium cepa root tips. Besides, thisaqueous extract of BPS inhibited the regeneration of blastema from amputated earthworms as well. Validation of this extract with MTT (3-(4,5-dimethyl thiazolyl-2-yl)- 2,5-diphenyltetrazolium bromide) assay using MCF-7 breast cancer cell linefurther supported the presence of antimitotic compounds. Aqueous BPS extract was further fractionated with ethyl acetateand it was found to inhibit the regeneration of new tissues from amputated earthworms. Liquid Chromatography and Mass spectrometry (LC-MS) analysis was performed with aqueousBPS extract to predict the lead compounds.Prediction analysis with mass values revealed the presence of three different compounds viz. α-tocotrienol, 1,2,4-nonadecanetriol and 3',4',7-trihydroxyisoflavone, which were already reported to inhibit the cell division. All our results strongly supported that banana pseudo-stem extract possesses antimitotic compounds. This is the first report of identification of putative antimitotic compounds from aqueous extract of Musa paradisiaca var. Robusta by using earthworms.
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