Cyperus Rotundus Extract Inhibits Acetylcholinesterase Activity From

Life Sciences 80 (2007) 2389–2392 www.elsevier.com/locate/lifescie

Cyperus rotundus extract inhibits acetylcholinesterase activity from animal and plants as well as inhibits germination and seedling growth in wheat and tomato ⁎ Rashmi Sharma, Rajendra Gupta

Department of Botany, University of Delhi, Delhi-110007, India Received 1 November 2006; accepted 26 January 2007

Abstract

Cyperus rotundus (nutgrass) is the world's worst invasive weed through tubers. Its success in dominating natural habitats depends on its ability to prevent herbivory, and to kill or suppress other plants growing in its vicinity. The present study was done to investigate whether chemicals in nutgrass target neuronal and non-neuronal acetylcholinesterases to affect surrounding animals and plants respectively. Methanolic extract of tubers of nutgrass strongly inhibited activity of AChE from electric eel, wheat and tomato. It also inhibited seed germination and seedling growth in wheat and tomato. Our results suggest that inhibitor of AChE in nutgrass possibly acts as agent of plant's war against (a) herbivore animals, and (b) other plants trying to grow in the same habitat. An antiAChE from nutgrass has been purified by employing chromatography and crystallization. The structural determination of the purified inhibitor is in progress. © 2007 Elsevier Inc. All rights reserved.

Keywords: Acetylcholinesterase; Allelopathy; Anticholinesterases; Cyperus rotundus; Nutgrass; Tomato; Wheat

Introduction investigation into the involvement of phytochemicals, as well as neuronal and non-neuronal acetylcholinesterase (AChE) in Acetylcholine (ACh) is a universal cell molecule in all plants mechanism of invasive success of a plant. We have probed Cy- and animals and is used in neuronal as well as non-neuronal perus rotundus (nutgrass), a weed in over 90 countries and signaling (Wessler et al., 2001). Plants have developed a repertoire considered to be world's worst weed through tubers (Holm et al., of toxic compounds that inhibit the cholinergic signal transduction 1977). (Roshchina, 2001). This has helped the plants to survive on earth during evolution even in the overwhelming presence of herbivores. Materials and methods Plants also compete with other plant species in their habitat by releasing allelochemicals that are harmful to the growth of other Plant material plants, a phenomenon known as allelopathy (Inderjit and Duke, 2003). The biochemical mechanism of some plant allelochemicals C. rotundus Linn. plants were collected from the botanical as a factor in invasiveness of weeds has been studied (Weston and gardens of the Department of Botany, University of Delhi, and Duke, 2003; Bais et al., 2003). However, it is surprising that its tubers were air-dried in shade for 10 days. Seeds of Lyco- ecological success and invasiveness of weeds has not been probed persicon esculentum Mill. (tomato) and Triticum aestivum Linn. for: (a) the possible involvement of ubiquitous signally molecule (wheat) were grown in Petri plates lined with wet filter paper ACh and (b) effect of plant cholinergic chemicals on animals. To (Whatman No. 1). the best of our knowledge, this paper presents the first ever Preparation of plant extract

⁎ Corresponding author. Tel.: +91 9212204551, +91 11 65460096. Tubers of C. rotundus were pulverised by grinding in liquid E-mail address: [email protected] (R. Gupta). nitrogen. Powdered tubers were extracted in methanol (1:5 w/v)

enzyme activity was stopped after 30 min by addition of neostigmine bromide to final concentration of 0.1 mM. Leaves of tomato (8-d-old) and shoots of wheat (5-d-old) were used as source of plant AChE. Finely chopped (2 mm pieces) of plant material were used for in vivo assay of AChE (Gupta and Gupta, 1997).

Purification of anticholinesterase from C. rotundus

The crude methanolic extract of tubers of C. rotundus was concentrated in vacuum to obtain a gummy mass. It was diluted in methanol and loaded on preparative thin layer chromatography plates (TLC). The TLC plates (20×15 cm) coated with Silica Gel G (0.5 mm thickness) were developed in ChCl3–MeOH (1:5 v/v). All portions of the TLC plates were tested for antiAChE activity; the active portion was eluted in MeOH and the procedure was repeated twice. The active band was eluted in methanol and crystallized.

Results

Methanolic extract of C. rotundus strongly inhibited AChE from electric eel in a concentration-dependent manner (Fig. 1A). A solution containing 0.5 mg Cyperus per ml caused 50% of the maximum inhibition. The Cyperus extract also inhibited AChE in leaves of wheat and tomato. The AChE in wheat was inhibited most followed by the enzyme from eel and tomato respectively (Fig. 1B). The inhibitory properties were intact even on heating the extract up to 60 °C in water bath for 45 min. The active Fig. 1. A. Effect of methanolic extract of Cyperus rotundus on electric eel compound has been crystallized and its chemical characterization AChE. B. Comparison of inhibition by Cyperus extract of AChE from eel, is in progress. The nutgrass extract inhibited seed germination, as tomato and wheat. Cyperus extract 3 mg/ml was used for each assay. The data well as growth of root and shoot in wheat and tomato (Fig. 2A–F). represents averages of three experiments and the bars represent ± S.E.M. Discussion and the extract was kept overnight before filtering through Whatman No. 1 filter paper. Plants and animals have co-evolved over millions of years and possess large a number of common biochemical mechanisms. Seed germination Ecological success of any plant species in nature depends on its ability to inhibit one or more crucial biochemical pathways in other Tomato and wheat seeds were placed in Petri plates (25× organisms so as to prevent being eaten by animals, and to prevent 150 mm and 15×90 mm for wheat and tomato respectively) lined nutritional competition from other plants in the same habitat. with filter paper (Whatman No. 1) soaked in different concentra- Several chemical compounds have been isolated from world's tion of Cyperus extract or solvents. One hundred seeds per Petri worst weed C. rotundus (Jeong et al., 2000; Sonwa and Konig, plate were placed almost equidistantly. 2001) and some of these chemicals possess medicinal properties (Singh et al., 1970; Gupta et al., 1971; Weenen et al., 1990; Acetylcholinesterase assay Hamada, 1993; Thebtaranonth et al., 1995; Makino et al., 2003). However, this paper reports for the first time the presence of AntiAChE activity on electric eel AChE (EC 3.1.1.7) was antiAChE properties in C. rotundus. Naturally, the presence of measured by employing a minor modification of Ellman's antiAChE in a plant would be a strong survival tool because it spectrophotometric assay (Ellman et al., 1961). The reaction would jeopardize herbivores' vital body functions of voluntary medium (4 ml) contained 0.1 M potassium phosphate buffer muscles as well as of autonomic nervous system. It is interesting to (pH 8), 0.1 mM dithiobis, 2-nitrobenzoate, 1 mM acetylthio- note that various preparations of C. rotundus have been used for cholne iodide, AChE from electric eel (Sigma–Aldrich, St. centuries in perfumes, spices and traditional medicines in India, Louis, USA) and 200 μl of different concentrations of the plant China, Arab and Africa. C. rotundus is also an important extract. Concentration of AChE was adjusted to give an ingredient of anti-aging Ayurvedic neutraceutical Chyavanprash. absorbance change of 0.8–1.0 in controls. Each assay was The presence of AChE in wheat and tomato is already reported done in triplicate. Experiments were carried out at 30 °C. The (Tretyn et al., 1986; Fletcher et al., 2004). Our results that R. Sharma, R. Gupta / Life Sciences 80 (2007) 2389–2392 2391

Fig. 2. Effect of methanolic extract of Cyperus rotundus on germination and seedling growth in wheat (A–C) and tomato (D–F). Observations on wheat were taken after 24 h of soaking for germination and after 72 h for root and shoot length. Observations on tomato were taken after 96 h of soaking for germination and after 120 h for root and shoot length. The data represents averages of three experiments and the bars represent ± S.E.M.

antiAChE present in Cyperus inhibits germination and growth in Prof. P. Pardha Saradhi for the use of Pharmacia Biopia-2000 wheat and tomato open interesting possibilities for further spectrophotometer. understanding the biochemical mechanism of allelopathy and ecological success of plants. References Conclusion Bais, H.P., Vepachedu, R., Gilroy, S., Callaway, R., Vivanco, J.M., 2003. Allelopathy and exotic plant invasion: from molecules and genes to species C. rotundus (nutgrass) contains a strong inhibitor of AChE, interactions. Science 301 (5638), 1377–1380. which possibly acts as an agent of plant's war against herbivore Ellman, G.L., Courtney, K.D., Andres Jr., V., Featherstone, R.M., 1961. A new animals, and other plants trying to grow in the same habitat. and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7 (7), 88–95. Acknowledgements Fletcher, S.P., Geyer, B.C., Smith, A., Evron, T., Joshi, L., Soreq, H., Mor, T.S., 2004. Tissue distribution of cholinesterases and anticholinesterases in native and transgenic tomato plants. Plant Molecular Biology 55 (1), 33–43. RS is grateful to the Council of Scientific and Industrial Gupta, A., Gupta, R., 1997. A survey of plants for presence of cholinesterase Research (CSIR) for Senior Research Fellowship and to activity. Phytochemistry 46 (5), 827–831. 2392 R. Sharma, R. Gupta / Life Sciences 80 (2007) 2389–2392

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