Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIII, 2012

THE EFFECT OF TRIFOLII RUBRI FLOS (RED CLOVER FLOWERS) HYDROALCOHOLIC EXTRACT ON SOME BIOCHEMICAL PARAMETERS IN TRITICUM AESTIVUM L. PLANTS
RUXANDRA CREU1*, LCRMIOARA OPRIC2, GABRIELA VOCHIA3, ELENA TRU3, CSILLA IULIANA BRA2, GOGU GHIORGHI4
Keywords: wheat, red clover, oxidoreductases, proteins. Abstract. Red clover (Trifolium pratense) hydroalcoholic extract was prepared by the extraction of powdered dried flowers with ethanol 70% v/v (1:13.33), by reflux for two hours. The stock extract was diluted with distilled water to give the final concentrations of 0.5, 1 and 5% (v/v). These concentrations were tested for their effects on superoxidedismutase, catalase and peroxidase activity in wheat (Triticum aestivum L.) seedlings, and on soluble protein content, in a laboratory experiment. Distilled water was used as a control (C). After the 7 days of experiment, we evaluated the activity of these oxidoreductases, as well as the soluble protein level.

INTRODUCTION
Traditionally, secondary metabolites in plants have been investigated by phytochemists. Originally classified as waste products, these compounds have recently been investigated extensively, and many complex biological functions have been discovered. Various secondary metabolites produced by plants and micro-organisms have been considered as potential allelochemicals (Abu-Romman et al., 2010; Ashrafi et al., 2009; Li et al., 2010; Yu et al., 2003). According to the different structures and properties of these compounds, allelochemicals can be classified into the following categories (Li et al., 2010): (1) watersoluble organic acids, straightchain alcohols, aliphatic aldehydes, and ketones; (2) simple unsaturated lactones; (3) longchain fatty acids and poly-acetylenes; (4) quinines; (5) phenolics; (6) cinnamic acid and its derivatives; (7) coumarins; (8) flavonoids; (9) tannins; (10) steroids and terpenoides (sesquiterpene lactones, diterpenes, and triterpenoids). Allelochemicals enter through the plant cell membrane and change the activity and function of certain enzymes. For example, chlorogenic acid, caffeic acid and catechol can inhibit phosphorylase activity; cinnamic acid and its derivatives can inhibit the hydrolysis activities of ATPase; tannic acids can inhibit activities of peroxidase (POD), catalase and cellulose (Li et al., 2010). Plants have the ability to fight against oxidative stress due to some intracellular defence strategies. These are represented by enzymatic (ascorbic acid, tocopherols, carotenoids, polyphenols, flavones, alkaloids) and non-enzymatic (superoxide-dismutase, catalase, peroxidase, ascorbate oxidase, glutathione reductase and polyphenol oxidase) antioxidant systems. Both systems act by preventing the induction or by elimination of free radicals generated during oxidative stress (Hum et al., 2006; Maxim et al., 2009; Mierlici et al., 2011; Olteanu et al., 2009, 2011). Trifolium pratense L. (red clover) contains (Barnes et al., 2007): estrogenic isoflavonoids (biochanin A, daidzein, formononetin and genistein); carbohydrates: arabinose, glucose, glucuronic acid, rhamnose, xylose (following hydrolysis of saponins glycosides), polysaccharide (a galactoglucomannan); flavonoids: isorhamnetin, kaempferol, quercetin, and their glycosides; saponins; coumaric acid; phaseolic acid; salicylic acid; vitamins and minerals. Red clover hydroalcoholic extract (70% v/v) contains tannins, reducing sugars, aminoacids, flavonoids, flavonoid glycosides, polyphenols, coumarins, and sterolic saponins (Cretu et al., 2011). The objective of this study was to determine the effects of red clover hydroalcoholic extract on enzymatic antioxidant activity, and soluble protein content in wheat (Triticum aestivum, Dropia cultivar) seedlings.

MATERIALS AND METHODS

Extracts preparation The fluid hydroalcoholic extract of Trifolium pratense (red clover) flowers (TPEx) was obtained from powdered dried material by reflux with 70% ethanol (1:13, 33), for two hours. The hydroalcoholic extract was filtered through a textile filter, and used as a stock extract. This was diluted with distilled water to give the final concentrations of 0.5, 1 and 5% (v/v) (TPEx1, TPEx2 and TPEx3). Seeds treatment Seeds of Triticum aestivum L. (Dropia cultivar from Secuieni Agricultural Research and Development Station, Neamt) were treated with different concentrations (0.5%, 1% and 5%) of red clover 70% hydroalcoholic extract, for 12

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Ruxandra Creu et al The effect of Trifolii rubri flos (red clover flowers) hydroalcoholic extract on some biochemical parameters in Triticum aestivum L. plants hours. Distilled water was used as a control (C). Seeds were washed with distilled water and placed in hydroponic system (constant level of water) and maintained for 7 days. Seedlings bioassay The biological material was represented by fresh shoots of 7 days old wheat seedlings. In order to analyze the enzyme activity, we carried out three samples (repetitions) for each experimental variant (extract concentration). The vegetal material was homogenized in chilled 0.1 M disodium phosphate buffer. The homogenate was centrifuged at 3000 rot/min for 15 min at 40C and the supernatant used for the determination of enzyme activity. Superoxide-dismutase (SOD) activity was determined spectrophotometrically at 560 nm, according to the method of Winterbourn, Hawkins, Brian and Carrell, adapted by Vlad Artenie (Artenie et al., 2008). Catalase (CAT) activity was assayed by Sinha method (Artenie et al., 2008), by measuring it spectrophotometrically at 570 nm. Peroxidase (POD) activity was determined with the method of L.V. Gudkova and R.G. Degtiari (Artenie et al., 2008), the increase in the absorbance being measured at 540 nm with a spectrophotometer. The results for each antioxidative enzyme were expressed in specific enzymatic units/mg protein. Protein determination was carried out according to Bradford method (Bradford, 1976). The extinction of reaction mixture was measured at 595 nm. Finally, the results were expressed in mg protein/g fresh weight. Statistical analysis The results were evaluated statistically by Students test. The means were compared by the least significant difference at p0.05.

RESULTS AND DISCUSSION Oxidative stress leads to the production of the reactive oxygen species (ROS) with severe disruptive effect upon the cellular metabolism (Olteanu et al., 2009) and the development of some processes that are often assigned to the alteration of the pattern of gene expression in living organisms (Mierlici et al., 2011). Recent studies suggest that environmental stress can increase the oxygen-induced damage to cells due to increased generation of reactive oxygen species (Niakan and Saberi, 2009; Yu et al., 2003). Thus, ROS has been proposed as a central component of plant response to biotic and abiotic stresses (Hammond-Kosack and Jones, 2000; Agarwal and Pandey, 2004) (cited by Hui-Qiong et al., 2006). In plants, antioxidant enzymes (e.g. superoxide-dismutase, peroxidase and catalase) act as a defensive system, which is involved in protecting cells from the injuries caused by the oxidative stress (Mittler, 2002-cited by Mierlici et al., 2011; Olteanu et al., 2009). The increase of the activity of these enzymes represents the most common pathway that leads to the elimination of ROS (Mierlici et al., 2011). 1. Effects on antioxidant enzymes activity in wheat seedlings after treatments with red clover extracts Superoxide-dismutase (SOD) works by converting the superoxide radical in hydrogen peroxide and oxygen by so called dismutation reaction (Olteanu et al., 2009, 2011). Because of the fact that SOD is present in all aerobic organisms and in almost all subcellular compartments that generate ROS, it is considered the most important for the protection against oxidative stress (Maxim et al., 2009). The principal scavenger of so formed H2O2 is CAT and, in certain situations, POD. The increase of CAT activity constitutes an indicator of a toxic accumulation of H2O2 which becomes inhibitory for SOD activity. Thus, SOD-CAT tandem serves as a front-line antioxidant defence (Olteanu et al., 2009). The effect of red clover extract at different concentrations on SOD activity in wheat seedlings is shown in Table 1. The mean value of SOD activity measured in control variant is equal to 4.302 USOD/mg protein. Relative to this, maximum concentration of the red clover extract induced an increase of SOD activity with 23.45%, which can indicate a possible oxidative stress expressed by the increase of superoxide radicals (O2-) concentration in the tissues of wheat seedlings. On the other hand, extract concentrations of 0.5 and 1% determined the reduction of this oxidative enzyme activity, with about 22.32% and 16.71%, respectively (3.342 and 3.583

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Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIII, 2012

USOD/mg protein), compared to control value. Thus, the three concentrations of red clover extract had different effects on SOD activity. Table 1. Effect of red clover extract on superoxide-dismutase (SOD) activity in the shoots of wheat seedlings SOD Mean activity Experimental (USOD/mg protein)* variants CV% t p (%) xSE Control (C) 4.302 0.384 15.451 TPEx1 (0, 5%) 3.342 0.052 2.679 2.4789 =0.0341 -22.32 TPEx2 (1%) 3.583 0.267 12.929 1.5379 >0.05 -16.71 TPEx3 (5%) 5.311 0.301 9.805 2.0690 >0.05 +23.45
*each value is the mean of three replicates and expressed as meanSE (average standard error); CV=average variation coefficient; t=tstatistical; p= probability; =increase/decrease rate.

Catalase is another antioxidant enzyme, whose activity is very important in the oxidative stress by diminishing the hydrogen peroxide level (the most stable between ROS). Our results showed the followings (table 2): an increase of the catalase activity with 18.50% compared to control, in TPEx1 experimental variant (minimum concentration of 0.5%), suggesting the presence of important amounts of hydrogen peroxide in the analyzed plantlets; treatments with 1% and 5% extract concentrations determined a slightly depression of average CAT activity in wheat shoots, both with 5.96% (0.300 UC/mg protein) compared to those measured in control variant (0.319 UC/mg protein). Table 2. Effect of red clover extract on catalase (CAT) activity in the shoots of wheat seedlings CAT Mean activity Experimental (UC/mg protein)* CV% variants t p (%) xSE Control (C) 0.319 0.012 6.606 TPEx1 (0, 5%) 0.378 0.035 16.039 1.5927 >0.05 +18.50 TPEx2 (1%) 0.300 0.023 13.061 0.7488 >0.05 -5.96 TPEx3 (5%) 0.300 0.030 17.049 0.5943 >0.05 -5.96
*each value is the mean of three replicates and expressed as meanSE (average standard error); CV=average variation coefficient; t=tstatistical; p= probability; =increase/decrease rate.

Peroxidase plays an important role in detoxification processes in two ways: regulator of the electronic flow in cell respiration, and scavenger of free radicals. Compared to catalase that is stimulated by higher quantities of oxidative agents, peroxidase is induced by lower quantities of H2O2 (Mierlici et al., 2011). Red clover extract (TPEx1, TPEx2 and TPEx3) acted differently on POD activity (table 3): the minimum and the maximum concentrations of 0.5% and 5% determined a slightly increase with 3.71% (0.886 UP/mg protein) in the former case, and a significantly one

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Ruxandra Creu et al The effect of Trifolii rubri flos (red clover flowers) hydroalcoholic extract on some biochemical parameters in Triticum aestivum L. plants

(p=0.0118) with 35.81% (1,134 UP/mg protein) in the latter case, and TPEx2 induced the reduction of enzyme activity with 9, 94%, compared to control value (0,835 UP/mg protein). Table 3. Effect of red clover extract on peroxide (POD) activity in the shoots of wheat seedlings POD Mean activity Experimental (UP/mg protein)* variants CV% t p (%) xSE Control (C) 0.835 0.055 11.362 TPEx1 (0, 5%) 0.866 0.048 9.511 0.4294 >0.05 +3.71 TPEx2 (1%) 0.752 0.057 13.133 1.0377 >0.05 -9.94 TPEx3 (5%) 1.134 0.064 9.779 3.5592 =0.0118 +35.81
*each value is the mean of three replicates and expressed as meanSE (average standard error); CV=average variation coefficient; t=tstatistical; p= probability; =increase/decrease rate.

A stimulation of the activities of POD and SOD by exposure to environmental stress has been documented in Ramalina farinacea L. (Aach), Lycopersicon pennellii Correll (D`Arcy), and Chlorella vulgaris Beij (Malanga and Puntarulo, 1995; Shalata and Tal, 1998; Deltoro et al., 1999) (cited by Yu et al., 2003). Activity levels of SOD and POD showed progressive significant increases with increased allelochemicals, compared to controls. This is likely related to stress, because allelopathy causes stress (Kamal and Bano, 2009). In contrast, it has been reported decreased activities of these antioxidant enzymes in Evernia prunastri L. (Ach.) and Lycopersicon esculentum Mill. under environmental (Politycka, 1996; Shalata and Tal, 1998; Deltoro et al., 1999) (cited by Yu et al., 2003). The treatments with some purinic derivates have determined the intensification of peroxidase and catalase activity and decrease of superoxidedismutase activity in Nicotiana tabacum L. plantlets (Stoica and Artenie, 2008). The extract of lantana leaves increased SOD activity in water hyacinth plant, and the increase in degree of membrane peroxidation, while the activity of catalase was inhibited by the treatment with lantana extract (Zeng et al., 2006). Extract from sunflower leaves increased peroxidase and superoxidedismutase in wheat (Kamal and Bano, 2009). Comparing the experimental variants treated with TPEx (0.5, 1 and 5%), we can observe the followings: TPEx1 determined a decrease of SOD comparatively to control, but CAT and POD levels are superior to control corresponding values. This is probably as a result of hydrogen peroxide accumulation that becomes inhibitive for SOD and activate CAT and POD-based antioxidative defence mechanisms (Olteanu et al., 2009); in case of TPEx2 treated variant, all enzymes showed decreased activities relative to control; increase of extract concentration to 5% induced the stimulation of SOD activity (1.24 times) compared to control as a response to superoxide accumulation. Compared to this, catalase is low, and peroxidase is 1.36 times higher than control variant. This may confirm the existence of alternative mechanisms which are able to remove hydrogen peroxide (compensation for catalase reduced activity). One of these mechanisms is peroxidase that in certain conditions can metabolize H2O2 formed in reaction catalyzed by SOD in a first stage (Olteanu et al., 2009). The increase in the production of SOD without a subsequent elevation of catalase (in our case, TPEx3 variant) or peroxidase leads to the accumulation of hydrogen peroxide who is converted into hydroxyl radical, which is toxic (Olteanu et al., 2009).

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Analele tiinifice ale Universitii Alexandru Ioan Cuza, Seciunea Genetic i Biologie Molecular, TOM XIII, 2012

2. Protein content in wheat seedlings The treatments with various environmental factors induce a large scale of morphological, physiological and biochemical modifications. At molecular level, these effects are reflected in change of expression pattern of some gene coding, (proteins and other compounds) (Olteanu et al., 2009). In stress condition, production and accumulation of ROS induce protein structural modifications: site-specific aminoacid modifications, fragmentation of the peptide chain, aggregation of cross-linked reaction products, and altered electrical charge (Davies, 2003) (cited by Olteanu et al., 2011). Oxidation of specific aminoacids because of increased reaction rate of superoxide anions with side chains of aminoacids marks proteins for degradation by specific proteases and can lead to cross-linking and to an increased susceptibility to proteolysis (Babior, 1997; Davies, 2003; Scandalios, 2005; Cargnelutti et al., 2006; Rellnlvarez et al., 2006) (cited by Olteanu et al., 2009). Protein oxidation in this way is often accompanied by increase of soluble protein amount (Gonalves et al., 2007) (cited by Olteanu et al., 2009). The shoots of 7 days old wheat seedlings responded to TPEx1 (0.5%) and TPEx3 (5%) by a diminished protein synthesis (5.636 mg/g fresh weight, and 5.105 mg/g fresh weight, respectively), comparatively to control value (6.094 mg/g fresh weight) (table 4). The maximum reduction of soluble protein level in TPEx3 variant was about 16.23%. On the other hand, the medium extract concentration induced an increase with 12.62% of protein amount. Increases of soluble protein can be the consequence of de novo synthesis of some stress proteins of exposure to environmental factors (Olteanu et al., 2009). Extract of sunflower leaves determine the increase of protein content in wheat (Triticum aestivum L.) (Kamal and Bano, 2009). The aqueous leaf leachate of Euphorbia hierosolymitana was found to decrease the amount of protein content (Abu-Romman et al., 2010). Table 4. Effect of red clover extract on protein content in the shoots of wheat seedlings Soluble proteins mg/g Experimental fresh weight* variants CV% t p (%) xSE Control (C) 6.094 0.309 8.774 TPEx1 (0, 5%) 5.636 0.181 5.554 1.2800 >0.05 -7.52 TPEx2 (1%) 6.863 0.272 6.876 1.8685 >0.05 +12.62 TPEx3 (5%) 5.105 0.357 12.119 2.0940 >0.05 -16.23
*each value is the mean of three replicates and expressed as meanSE (average standard error); CV=average variation coefficient; t=tstatistical; p= probability; =increase/decrease rate.

CONCLUSIONS In our study, we have investigated the effects of a red clover - 70% hydroalcoholic extract at different concentrations on some biochemical parameters (antioxidant enzymes activity and protein content) in wheat plantlets. We have evaluated specific enzymatic activity of superoxidedismutase, catalase and peroxidase, and determined the level of soluble proteins. Our treatments showed some variations of SOD, CAT and POD activities, but not closely related to extract concentrations. Also, we have observed some variability differences between these oxido-reductases. Thus, SOD showed higher limits of variability at all concentrations, compared

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to CAT and POD which registered higher limits only at one concentration: minimum in the former case, and maximum in the latter. It was not established a direct correlation between red clover extract concentration and soluble protein level. REFERENCES
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1 The Commercial Society for Medicinal Plant Research and Processing PLANTAVORELS.A., Cuza-Voda 46, PiatraNeamt, Romania 2 University Alexandru Ioan Cuza, Faculty of Biology, Carol I Bd. 20A, Iasi, Romania 3 Biological Research Institute, Lascar Catargi 47, Iasi, Romania 4 Academy of Romanian Scientists; University Alexandru Ioan Cuza, Faculty of Biology, Carol I Bd. 20A, Iasi, Romania * ruxycretu@yahoo.com

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