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

THE EFFECT OF TRIFOLII RUBRI FLOS(RED CLOVER FLOWERS)

HYDROALCOHOLIC EXTRACT ON SOME BIOCHEMICAL

PARAMETERS IN TRITICUM AESTIVUML. 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 driedflowers with ethanol 70% v/v (1:13.33), by reflux for two hours. The stock extract was diluted with distilled water to givethe final concentrations of 0.5, 1 and 5% (v/v). These concentrations were tested for their effects on superoxide-dismutase, catalase and peroxidase activity in wheat (Triticum aestivum L.) seedlings, and on soluble protein content, in alaboratory experiment. Distilled water was used as a control (C). After the 7 days of experiment, we evaluated the activityof these oxidoreductases, as well as the soluble protein level.

INTRODUCTION

Traditionally, secondary metabolites in plants have been investigated by phytochemists. Originally classifiedas waste products, these compounds have recently been investigated extensively, and many complex biological functionshave 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 tothe 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) simpleunsaturated lactones; (3) longchain fatty acids and poly-acetylenes; (4) quinines; (5) phenolics; (6) cinnamic acid and itsderivatives; (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 certainenzymes. 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 duringoxidative stress (Humet 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 (followinghydrolysis 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 enzymaticantioxidant activity, and soluble protein content in wheat (Triticum aestivum,Dropiacultivar) seedlings.

MATERIALS AND METHODS

Extracts preparation

The fluid hydroalcoholic extract of Trifolium pratense (red clover) flowers (TPEx) was obtained frompowdered dried material by reflux with 70% ethanol (1:13, 33), for two hours. The hydroalcoholic extract was filteredthrough a textile filter, and used as a stock extract. This was diluted with distilled water to give the final concentrations of0.5, 1 and 5% (v/v) (TPEx1, TPEx2 and TPEx3).

Seeds treatment

Seeds of Triticum aestivumL. (Dropiacultivar 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 biochemicalparameters in Triticum aestivumL. 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 theenzyme 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 3000rot/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 andCarrell, adapted by Vlad Artenie (Artenie et al., 2008). Catalase(CAT) activity was assayed by Sinha method (Artenie etal., 2008), by measuring it spectrophotometrically at 570 nm. Peroxidase(POD) activity was determined with the methodof L.V. Gudkova and R.G. Degtiari (Artenie et al., 2008), the increase in the absorbance being measured at 540 nm with aspectrophotometer. 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 ofreaction mixture was measured at 595 nm. Finally, the results were expressed in mg protein/g fresh weight.

Statistical analysisThe 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) withsevere disruptive effect upon the cellular metabolism (Olteanu et al., 2009) and the developmentof some processes that are often assigned to the alteration of the pattern of gene expression inliving organisms (Mierlici et al., 2011). Recent studies suggest that environmental stress canincrease the oxygen-induced damage to cells due to increased generation of reactive oxygenspecies (Niakan and Saberi, 2009; Yu et al., 2003). Thus, ROS has been proposed as a centralcomponent 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) actas a defensive system, which is involved in protecting cells from the injuries caused by theoxidative stress (Mittler, 2002-cited by Mierlici et al., 2011; Olteanu et al., 2009). The increaseof the activity of these enzymes represents the most common pathway that leads to theelimination of ROS (Mierlici et al., 2011).

1. Effects on antioxidant enzymes activity in wheat seedlings after treatments with

red clover extractsSuperoxide-dismutase (SOD) works by converting the superoxide radical in hydrogen

peroxide and oxygen by so called dismutation reaction (Olteanu et al., 2009, 2011). Because ofthe fact that SOD is present in all aerobic organisms and in almost all subcellular compartmentsthat 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 certainsituations, POD. The increase of CAT activity constitutes an indicator of a toxic accumulation ofH2O2which becomes inhibitory for SOD activity. Thus, SOD-CAT tandem serves as a front-lineantioxidant defence (Olteanu et al., 2009).

The effect of red clover extract at different concentrations on SOD activity in wheatseedlings is shown in Table 1. The mean value of SOD activity measured in control variant isequal to 4.302 USOD/mg protein. Relative to this, maximum concentration of the red cloverextract induced an increase of SOD activity with 23.45%, which can indicate a possible oxidativestress expressed by the increase of superoxide radicals (O2-) concentration in the tissues of wheatseedlings. 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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Ruxandra Creu et al The effect of Trifolii rubri flos(red clover flowers) hydroalcoholic extract on some biochemicalparameters in Triticum aestivumL. plants

(p=0.0118) with 35.81% (1,134 UP/mg protein) in the latter case, and TPEx2 induced thereduction 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

Experimental

variants

POD

Mean activity

(UP/mg protein)* 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 +3.71TPEx2 (1%) 0.752 0.057 13.133 1.0377 >0.05 -9.94TPEx3 (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 hasbeen documented inRamalina farinaceaL. (Aach),Lycopersicon pennelliiCorrell (D`Arcy), andChlorella 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 significantincreases 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 reporteddecreased activities of these antioxidant enzymes in Evernia prunastri L. (Ach.) andLycopersicon 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 superoxide-dismutase activity in Nicotiana tabacumL. plantlets (Stoica and Artenie, 2008). The extract oflantana leaves increased SOD activity in water hyacinth plant, and the increase in degree ofmembrane peroxidation, while the activity of catalase was inhibited by the treatment with lantanaextract (Zeng et al., 2006). Extract from sunflower leaves increased peroxidase and superoxide-dismutase in wheat (Kamal and Bano, 2009).

Comparing the experimental variants treated with TPEx (0.5, 1 and 5%), we can observethe followings: TPEx1 determined a decrease of SOD comparatively to control, but CAT andPOD 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, allenzymes 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 tosuperoxide accumulation. Compared to this, catalase is low, and peroxidase is 1.36 times higherthan control variant. This may confirm the existence of alternative mechanisms which are able toremove hydrogen peroxide (compensation for catalase reduced activity). One of thesemechanisms is peroxidase that in certain conditions can metabolize H2O2 formed in reactioncatalyzed by SOD in a first stage (Olteanu et al., 2009). The increase in the production of SODwithout a subsequent elevation of catalase (in our case, TPEx3 variant) or peroxidase leads to theaccumulation 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 Genetici Biologie Molecular, TOM XIII, 2012

2. Protein content in wheat seedlings

The treatments with various environmental factors induce a large scale ofmorphological, physiological and biochemical modifications. At molecular level, these effectsare 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 ROSinduce protein structural modifications: site-specific aminoacid modifications, fragmentation ofthe 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 ofincreased reaction rate of superoxide anions with side chains of aminoacids marks proteins fordegradation by specific proteases and can lead to cross-linking and to an increased susceptibilityto proteolysis (Babior, 1997; Davies, 2003; Scandalios, 2005; Cargnelutti et al., 2006; Relln-lvarez et al., 2006) (cited by Olteanu et al., 2009). Protein oxidation in this way is oftenaccompanied by increase of soluble protein amount (Gonalves et al., 2007) (cited by Olteanu etal., 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 maximumreduction of soluble protein level in TPEx3 variant was about 16.23%. On the other hand, themedium extract concentration induced an increase with 12.62% of protein amount.

Increases of soluble protein can be the consequence of de novosynthesis of some stressproteins of exposure to environmental factors (Olteanu et al., 2009).

Extract of sunflower leaves determine the increase of protein content in wheat (TriticumaestivumL.) (Kamal and Bano, 2009). The aqueous leaf leachate of Euphorbia hierosolymitanawas 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

Experimental

variants

Soluble proteins

mg/g

fresh weight* 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 -7.52TPEx2 (1%) 6.863 0.272 6.876 1.8685 >0.05 +12.62TPEx3 (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 atdifferent concentrations on some biochemical parameters (antioxidant enzymes activity and

protein content) in wheat plantlets. We have evaluated specific enzymatic activity of superoxide-dismutase, catalase and peroxidase, and determined the level of soluble proteins.Our treatments showed some variations of SOD, CAT and POD activities, but not closely relatedto extract concentrations. Also, we have observed some variability differences between theseoxido-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 theformer case, and maximum in the latter.It was not established a direct correlation between red clover extract concentration and soluble

protein level.

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1The Commercial Society for Medicinal Plant Research and Processing PLANTAVORELS.A., Cuza-Voda 46, Piatra-Neamt, Romania2 University Alexandru Ioan Cuza, Faculty of Biology, Carol I Bd. 20A, Iasi, Romania3 Biological Research Institute, Lascar Catargi 47, Iasi, Romania4 Academy of Romanian Scientists; University Alexandru Ioan Cuza, Faculty of Biology, Carol I Bd. 20A, Iasi,Romania* ruxycretu@yahoo.com

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