Antigenotoxic Effects of Brassica oleracea L. Var. Italica Aqueous Seed Extract on Allium cepa Root Chromosomal Aberration Assay

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1 International Journal of Genetics 2(2): 22-28, 2012 ISSN IDOSI Publications, 2012 DOI: /idosi.ijg Antigenotoxic Effects of Brassica oleracea L. Var. Italica Aqueous Seed Extract on Allium cepa Root Chromosomal Aberration Assay Anita Kumari, Sonia Sharma and Adarsh Pal Vig Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar , Punjab, India Abstract: The herbicide cilluron containing cyhalofop-butyl (10%) is an active ingredient. Cyhalofop-butyl has low oral, dermal and inhalation toxicity. In the present study the antigenotoxic effects of Brassica oleracea L. var. italica was evaluated using Allium cepa root chromosomal aberration assay against ciluron. Three types of treatments were given to roots in pre-treatment roots were first treated with different concentrations (0.5, 1, 2, 4, 8 and 16 mg/ml) of extract for 3 hours followed by ciluron (0.8%). In post-treatment roots were first treated with ciluron followed by different concentrations of extract for 3 hours. In simultaneous treatment roots were treated with different concentrations of extract and ciluron simultaneously for 3 hours. The treatment of roots with distilled water and 0.8% ciluron served as negative and positive control. All the three types of treatments of extract showed dose dependent decrease in number of chromosomal aberrations. These results showed that Brassica oleracea L. var. italica aqueous seed extract have antigenotoxic effects. Key words: Ciluron % Brassica Oleracea % Allium Cepa % Chromosomal Aberration INTRODUCTION and in vivo. The A. cepa root chromosomal aberration assay was simple and reliable as the method where To increase the agricultural production, pesticides are chromosomal aberrations were recorded in all types of some of the compounds most frequently released into the mitotic cells [7]. environment. Nowadays more than 1500 chemicals are In this study, Allium cepa root chromosomal registered for use in formulation of pesticides and there aberration assay was used in the evaluaton of are approximately 600 pesticides active ingredient in antigenotoxic activity of water extract of seeds of broccoli market [1-2]. Despite the beneficial effects associated with against the genotoxicity of herbicide ciluron. Cilluron the use of pesticides, many of these chemicals also pose containing cyhalofop-butyl (10%) is an active ingredient. hazards to human life and environment [3]. In spite of the Cyhalofop-butyl is an aryloxyphenoxy propionate inconvience, it is not possible to reduce the pesticides herbicide for post emergence control of barnyard grasses without reducing crop yield [4]. Alternative methods need and silver top grass in rice. Cyhalofop-butyl has low oral, long period to be effective and the results are not dermal and inhalation toxicity. It is known to induce liver completely satisfactory, therefore, release of pesticides and kidney abnormalities in experimental animals. into the environment is continuously increasing day by The medicinal plants have assumed increasing day. For this reason, it is necessary to extend the importance as a very useful therapeutic alternative in the evaluation of the genotoxicity of the chemicals. The programs of primary health care. Plants have been utilized genotoxic effects of many pesticides have been evaluated as medicines from thousands of years [8]. The World by several researchers through various test systems [5-6]. Health Organization (WHO) has recognized, the Various genotoxic assays are known over the past importance of herbal medicine, suggesting that it is a years can be used to study the genotoxicity and viable alternative and important also for people in antigenotoxicity in a variety of methods. Since the results developing countries, since its cost is decreased. A large of various studies using Allium cepa fit best in test number of natural products referred as chemo preventive battery composed of prokaryotes and eukaryotes in vitro agents that offer a greater effect against cancer through a Corresponding Author: Adarsh Pal Vig, Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar , Punjab, India. 22

2 range of mechanisms including antioxidant, antimutagenic defatted with hexane to remove fatty oil etc. After activity, enzyme modulation, gene expression, apoptosis filtration, seed meal was air dried for overnight and etc. [9-11] extracted with double amount of water by placing the Cruciferous vegetables particularly the members of mixture on shaker for two nights. Solution was filtered Brassica genus such as broccoli, cauliflower, cabbage with whatmann s filter paper. Water was distilled-off to are rich source of glucosinolates which on enzymatic concentrate the water extract and the extract was dried by degradation breakdown into various hydrolytic products evaporating on water bath. Different concentrations were like isothiocyanates, nitriles etc. Among all, prepared by dissolving per mg of each extract first in isothiocyanates are protective against the most common dimethyl sulfoxide (DMSO) and then equal amount of cancer types such as lung, prostrate, breast and colon water was added to have final composition of mg/ml. cancers [12-14]. Broccoli contains a wide range of Afterwards different concentrations were prepared (0.5, 1, phytochemicals, including glucosinolates, flavonols and 2, 4, 8 and 16 mg/ml) from the stock solution. carotenoids. It also contains high concentrations of selenium and glucosinolates, particularly glucoraphanin Allium cepa Root Growth Test/ Determination of EC 50: and isothiocyanate sulforaphane [15] which have anticarcinogenic properties [16]. Brassica oleracea L. var. italica gave protection against cancer [17] and heart disease [18]. Antioxidant activity of broccoli on cooked food and juices has also been reported [19, 20]. From the epidemiological data it was suggested that diet rich in Brassica vegetables such as Broccoli (Brassica oleracea var. italica) are associated with a lower risk of lung and Clean and healthy onion bulbs were set up and allowed to germinate in distilled water for 24 hours, where after the homogenously rooted bulbs were selected for control and different concentrations (2, 1, 0.8, 0.6, 0.4, 02 and 0.1% ) of ciluron herbicide for 96 hours. During the experiment, the test solutions were changed after every 24 hours. The root lengths from the control and experimental sets were measured (lengths of ten roots from each bulb) at the end colorectal cancer. 56% of the case controlled studies of exposure time. In this experiment Effective demonstrate a strong relationship between increased Concentration (EC 50) refers to be the effective broccoli consumption and the protection against cancer [21-23]. concentration which indicates the 50% root growth inhibition. In this study, we report antigenotoxic effects of aqueous extract of seeds of broccoli on cell division in root tips against ciluron herbicide using Allium cepa root chromosomal aberration assay. MATERIALS AND METHODS Test Organism: Equal sized bulbs of a commercial variety of Alllium cepa L. were taken from the local market. The outer scales of the bulbs were carefully removed and the brownish bottom plates were scrapped away without destroying the root primordia. Test Chemicals: Ciluron (Cyhalofop butyl) herbicide was taken from local market Hall Gate, Amritsar, Punjab. Orcein, glacial acetic acid, HCl and other chemicals were bought from Qualigens Fine Chemicals, Mumbai, India, Thomas Baker (Chemicals) PVT, Limited, Mumbai, India. Seeds Collection and Extraction: Certified seeds of Brassica oleracea L. var. italica (Broccoli) were procured from Chaudary Sarvan Kumar Agriculture University, Palampur, Himachal Pradesh, India. They were grinded to a fine powder. The finely ground powder of seeds were Antigenotoxic Effect of Aqueous Seed Extract: Antigenotoxic effects of aqueous extract of seeds of Broccoli were estimated by analyzing genotoxicity induced by ciluron herbicide in root tip meristematic cells of Allium cepa. The loose outer scales were removed and the brown roots were scrapped to expose the primordia. Onion bulbs were grown on coupling jars containing distilled water until roots grew up to the average length of cm. Three modes of treatment were given to roots. In pre-treatment roots were first treated with different concentrations (0.5, 1, 2, 4, 8 and 16 mg/ml) of extract for 3 hours followed by ciluron treatment (0.8%, 3 hours). In post-treatment, roots were first treated with ciluron followed by different concentrations of extract for three hours. In simultaneous treatment roots tips were treated with ciluron (0.8%) and different concentration of extract simultaneously for 3 hours. The treatment of roots with 0.8% of ciluron and distilled water served as positive and negative control. After treatment, the bulbs were washed under tap water and root tips were removed from the bulb, fixed in Farmer s fluid (glacial acetic acid and ethyl alcohol 1:3) for 24 hours. The slides were prepared by hydrolyzing the root tips in 1N HCl with intermittent heating for 1 23

3 minute and then squashed in aceto-orcein and 1N HCl RESULTS (9:1). The tip of root was cut and placed on slide, covered with cover slip and squashed by match stick and sealed The average roots length for control and treatment with DPX. Each slide was examined under the microscope groups is given in Fig. 1. The effect of aqueous extract of and photographed using Nikon camera. Different kinds of broccoli which was applied before, after and chromosomal aberrations were scored. About 600 dividing simultaneously ciluron treatment on root length is cells from 9-10 root tips were scored for each treatment. significant when compared with positive and negative The percentage inhibition (PI) of chromosomal aberrations controls. The measures of average root length in negative was calculated as follows: PI = a-b/a-c x 100 where, a is the and positive control are 3.3 cm and 2 cm. However it is number of aberrant cells induced by positive control clear from the result that roots length increase as the (0.8% ciluron), b is the number of aberrant cells induced concentration of extract increase from 0.5 to 16 mg/ml by seed extract and ciluron (0.8%) and c is the number of (Fig. 1). aberrant cells induced by negative control (distilled Firstly the genotoxic effect of different water). concentrations (0.1, 0. 2, 0.4, 0.6, 0.8 and 1%) of ciluron was evaluated. Among different concentrations, 0.8% Statistical Analysis: The experimental data is presented concentration of ciluron was the effective concentration as mean±se of triplicate experiment. The linear (EC 50) and was further used in antigenotoxic studies. relationship between dose and effects of aqueous extract Ciluron induced various types of chromosomal was obtained by simple regression and correlation aberrations partioned into physiological aberrations analysis. (17.6%) like c-mitosis, delayed anaphase, laggards etc. Fig 1: Average roots length in control and different concentrations of aqueous extract of Brassica oleracea L. var. italica. NC= Negative control (distilled water); PC= Positive Control (ciluron: 0.8 %) Table 1: Effect of pre-, post-and simultaneous-treatments of aqueous extract of Broccoli seeds on genotoxic effects induced by ciluron herbicide in root tip cells of Allium cepa 0.5mg/ml 1 mg/ml 2 mg/ml 4mg/ml 8mg/ml 16mg/ml Negative Positive control No. of No. of No. of No. of No. of No. of Type of control (ciluron-0.8%) Aberrant cells Aberrant cells Aberrant cells Aberrant cells Aberrant cells Aberrant cells a a a a a a chromosomal No. of No. of a a aberrations Aberrant cells Aberrant cells A B C A B C A B C A B C A B C A B C Physiological aberrations (PA) C-mitosis Delayed anaphase Laggard/s Stickiness Vagrant/s Mutipolarity Total PA Clastogenic aberrations (CA) Chromatin bridge/s Chromosomal break/s Total CA Total aberrant cells (PA+CA) a= out of 600 cells examined; A= Pre-, B= Post-and C= Simultaneous-treatment 24

4 Intl. J. Genet., 2(2): 22-28, 2012 Fig. 2: Root tip cells of Allium cepa showing physiological and clastogenic abbreations: a) vagrant chromosome b) chromatid bridge c) c-mitosis d) chromosomal break e) multipolarity f) stickiness and clastogenic aberrations (2.3%) like chromatin bridge/s, chromosal break/-s (Fig. 2). It induces more physiological aberrations than clastogenic aberrations. Cmitosis followed by delayed anaphase was the most frequent kind of aberrations found in dividing cells. The effects of pre-, post-and simultaneous treatment of aqueous extract of seeds of broccoli resulted in a dose dependent decrease in chromosomal aberrations (Table 1). All the three types of treatment of extract were found to be equally effective. Post and simultaneous treatments of aqueous extract of broccoli were found to be very effective and resulted in a significant decrease in clastogenic aberrations (100% at the highest dose tested). On other hand pre-treatment of extract cause significant reduction in physiological aberrations (98.9% at the highest dose tested). Among the three treatments 25

5 Fig. 3: Relationship between different concentrations (0.5, 1, 2, 4, 8 and 16 mg/ml) of water extract of B. oleracea L. var. italica seeds and percent inhibition of genotoxic effects induced by ciluron herbicide (0.8%) in Allium cepa root chromosomal aberration assay. simultaneous treatment showed maximum percentage Brassica oleracea L. var. italica (broccoli) is one of the inhibition (98.9%) followed by pre-treatment (97.9%) and major agricultural products widely considered to contain post-treatment (95.8%). The linear regression analysis high level of phytochemicals including glucosinolates, 2 method of determining the P value (R ) indicates that the flavonoids, vitamins and minerals [27, 28]. Many studies percent inhibition of chromosomal aberration was dose have indicated that consumption of broccoli is inversely dependent and positively correlated (Fig. 3). related with the occurrence of cancer in human. The health benefits of vegetables in preventing cancer and DISCUSSION cardiovascular diseases are mostly attributed to the quality of antioxidative components. The main In this study, the effect of different antioxidative components present in broccoli are concentrations of the aqueous extract of Broccoli flavonoids and vitamins. Broccoli sprouts have also been seeds were observed on genotoxicity of ciluron reported to inhibit cancer growth in vitro and in vivo to employing Allium cepa root chromosomal aberration extend their protective role against cancer [29]. Dietary assay. In this study, ciluron was observed to cause dose broccoli sprouts and their component glucosinolates and dependent decrease in root length and increase different isothiocyanates induce phase II enzymes and afford types of chromosomal aberrations with increase in protection against chemically induced tumours in rodents concentrations. [30, 31]. Plants have been used as an indicator organism in Allium cepa root chromosomal aberration assay is studies on mutagenesis in higher eukaryotes. Plant being used to determine the antigenotoxicity of different systems have a variety of well defined genetic endpoints plant extracts Terminalia chebula [32], Ocimum sanctum like structural alterations in ploidy, chromosomal L. leaf [33], Plantago lanceolata [34], Curcumin [35], aberrations and sister chromatid exchanges (SCE) [24-26] Aegle marmelos [36], bitter leaf [37], Brassica juncea [7]. 26

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