ANTIOXIDANT AND ANTIPROTEINASE EFFECTS OF BUCKWHEAT HULL EXTRACTS

Buckwheat is known not only due to its appropriate nutritional composition but the content of prophylactic compounds, too. These are responsible for buckwheat beneficial impact on human health. Most of them are concentrated in outer layers of buckwheat grain. The subject of this work was to screen hulls of nine common and one tartary buckwheat cultivar for the content of flavonoids and its antioxidant and antiproteinase effects. The highest content of total flavonoids was determined for tartary buckwheat cultivar Madawaska (0.6% of hulls weight). Among common buckwheat cultivars the best values reached samples Bamby (0.23%) and KASHO-2 (0.11%). Antioxidant activity as detected via binding radical ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and monitoring reducing power was the most effective for samples with highest flavonoid content. Buckwheat hulls effectively inhibited pathophysiological proteases thrombin and urokinase, whereas only little effects were seen to trypsin and elastase. In this testing there were again the best samples with highest flavonoid content. Only tartary buckwheat Madawaska effectively inhibited elastase at tested concentrations. No significant correlation was determined between flavonoid content and measured antioxidant or protease inhibitory action. Obtained results allow us to commend tartary buckwheat cultivar Madawaska as well as common buckwheat cultivars Bamby and KASHO-2 for further experiments.


INTRODUCTION
Food is not only a source of energy and nutrition for maintenance and growth of the body but is also a source of bioactive compounds that have beneficial effects on humans.Common buckwheat (Fagopyrum esculentum) and tartary buckwheat (Fagopyrum tataricum) are traditional foodstuffs available worldwide.Buckwheat is a traditional crop in Central and Eastern Europe and Asia.It is not a cereal, but buckwheat seed has chemical, structural and utilisation characteristics similar to those of cereal grains and thus is usually classified as a pseudocereal (Yildiz, Bilgiçli, 2012).
Buckwheat represents raw material interesting in term of its health beneficial properties.It contains many prophylactic compounds that can in the positive way influence genesis and development of many diseases.Dietary fibre is contained mainly in coating layers.It is usefull in preventing gastrointestinal disorders.Phytosterols present in endosperm lower blood cholesterol.Buckwheat bran is rich in B group vitamins.Due to binding proteins they are more bioavailable than from other sources.In comparison with other cereals and pseudocereals buckwheat is better source of magnesium, potassium, phosphorus, zinc, manganese and copper.They are located in peripheral layers and in embryo (Danihelová, Šturdík, 2012).
Buckwheat is known as one of the richest sources of polyphenols and flavonoids.These are concentrated mainly in outer layers of buckwheat grain (Sedej et al., 2012).Among them the most abundant is rutin with its content from 0.02% to 2% (Jiang et al., 2007).In buckwheat we can found also other polyphenolssinapic, ferulic, syringic or protocatechuic acid This paper links to the previous one, that was aimed at screening of buckwheat cultivars for their cytotoxic and antioxidant activity (Danihelová, Jantová, Šturdík, 2013).The subject of this work was to screen nine common buckwheat cultivars and one tartary buckwheat cultivar for total flavonoid content.We have tested buckwheat hull methanolic extracts.Samples were examined for antioxidant activity as detected via binding radical ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6sulphonic acid)) and via measuring reducing power Volume 7 90 No. 1/2013 (FRAP).Inhibitory activity to serine proteases trypsin, thrombin, urokinase and elastase was also determined.

PLANT MATERIAL TREATMENT
Nine common buckwheat cultivars and one tartary buckwheat cultivar were kindly provided from Plant production research center in Piešťany (SR).Overview of tested cultivars is outlined in Table 1.Buckwheat grains were mechanically dehulled.Obtained hulls were extracted using methanol (p.a.) for 24 hours at room temperature (diluent : weighing material = 10 : 1), filtered and used for flavonoid content determination and antioxidant activity testing.For purposes of enzyme inhibition evaluation extracts were evaporated and dissolved in dimethyl sulfoxide.2002).The 200 µl of 5% AlCl 3 methanolic solution was added to 2 ml of sample.After 30 min flavonoid-aluminium complex was detected via measuring absorbance at 420 nm.Samples were measured in three replicates.Standard curve of rutin was prepared using the similar procedure.Results were expressed in rutin equivalents (mg RE/g dry sample).Data were presented as means of the percentage of control ± SD (standard deviation).

FREE RADICAL SCAVENGING ACTIVITY (ABTS)
The ability to scavenge free radicals was observed using spectrophotometric method according Re et al. (1999).Cationradical ABTS ˙+ was prepared by reaction between 7 mM ABTS in phosphate buffered saline (0,1 M, pH 7.4) and 2.45 mM potassium persulfate in phosphate buffered saline (0.1 M, pH 7.4) in the rate of 1:1.This mixture stayed at room temperature in the dark for 12 hours.Solution of cationradical ABTS ˙+ was diluted with methanol (1.5 ml of ABTS ˙+ was pipetted into 60 ml of methanol) to get an absorbance of 0.700 at 734 nm.Then 1.95 ml of diluted ABTS ˙+ was added to 0.05 ml of sample.Reaction mixture was incubated 7 min at room temperature in the dark.Thereafter the absorbance was measured at 734 nm.Samples were measured in three replicates.Trolox served as standard antioxidant control.Results were expressed in trolox equivalents (µM TE/g dry sample).Data were presented as means of the percentage of control ± SD (standard deviation).

FERRIC REDUCING ANTIOXIDANT POWER (FRAP)
Antioxidant reducing power of tested samples was performed using FRAP method according Benzie and Strain (1996).This spectrophotometric procedure measures the ability to reduce ferric complex to ferrous.Working FRAP reagent was prepared by mixing 10 ml of acetate buffer (0.1 M, pH = 3.6), 2.5 ml of 10 mM TPTZ (2,4,6-tris(2-pyridyl)-S-triazine) in 40 mM HCl and 2.5 ml of 20 mM FeCl 3 •6 H 2 O. 25 µl of sample and 175 µl of FRAP reagent were pipetted into microplate well.The reaction lasted for 10 min at 37 °C.At the end, absorbance changes were measured spectrophotometrically at 593 nm.Samples were measured in five replicates.Trolox served as standard antioxidant control.Results were expressed in trolox equivalents (µM TE/g dry sample).Data were presented as means of the percentage of control ± SD (standard deviation).
All tested extracts were initially solubilized in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/ml and subsequently diluted in the reaction mixture to final concentrations 6.25 -100 µg/ml.The highest concentration of DMSO in the reaction mixture never exceeded 2 %.The absorbance was measured in the 1 st and 61 st minute after reaction started.Each experiment was performed in quintuplicate.Inhibitory activity was expressed as the concentration that is responsible for 50 % of substrate cleavage inhibition (IC 50 ).Data were presented as means of the percentage of control ± SD (standard deviation).

TOTAL FLAVONOID CONTENT
Among natural plant sources rich in bioactive compounds we have choosed buckwheat due to its high content of rutin, tradition of cultivation in Slovakia as well as large scale of documented biological effects (Krkošková, Mrázová, 2005).Buckwheat hulls represent waste material that has no important commercial utilization.But in comparison with other parts of buckwheat grain in hulls are concentrated present polyphenols and flavonoids (Sedej et al., 2012).We therefore decided to use these in our experiments.From the collection of nine common buckwheat cultivars and one tartary buckwheat cultivar we prepared hull extracts in methanol using diluent to weighing material ratio 10:1.In samples we first determined total flavonoid content.Results are presented in Figure 1.According to the literature the highest flavonoid content was detected in tartary buckwheat (about 0.6% of hulls weight).The most of common buckwheat cultivars have shown approximately 10 times lower content of flavonoids as compared to tartary buckwheat.Among common buckwheat cultivars the most flavonoids contained cultivars KASHO-2 (0.11%) and Bamby (0.23%).

Sedej et al. (2012)
found significantly higher content of total flavonoids in buckwheat hull than in whole grain and groat.Also other authors discovered that flavonoids are more abundant in hulls than in the flour (45.6 mg/100 g DW for hulls and 9.8 mg/100 g DW for flour) (Quettier- Deleu et al., 2000).
Obtained data from the literature about buckwheat flavonoid content lie in the wide range because they are dependent on varietal and growth conditions.Common buckwheat hulls contained total flavonoids from 36 mg/100 g to 1180 mg/100 g of hulls weight (Watanabe, Ohshita, Tsushida, 1997;Quettier-Deleu et al., 2000;Sedej et al., 2008).Data stated for tartary buckwheat hulls are higher -1100 mg/100 g to 3000 mg/100 g of hulls weight (Yongyan et al., 2007;Xiong et al., 2009).Our results are comparable with these values, but flavonoid content for tartary buckwheat is lower.

ANTIOXIDANT ACTIVITY
Because flavonoids are known for their antioxidant properties and our previous investigations have shown, that buckwheat hull extracts possess antioxidant action, in the next step we have examined their antioxidant activity using other two different spectrophotometric methods.The first one follows the ability to bind cationradical ABTS ˙+.Antioxidants present in buckwheat caused radical binding and thereby its decolorization.The second one measures reducing power of samples (FRAP).Active samples could reduce ferric complex to ferrous, what resulted in color change.Activity was compared to standard antioxidant trolox (TE = trolox equivalent).Determined effects are presented in Table 2. Comparing obtained results of both measurements we came to the same conclusion.Antioxidant activity determined via binding radical ABTS as well as measuring reducing power (FRAP) was the highest in the case of tartary buckwheat Madawaska, which concurrent contained the highest amount of total flavonoids among tested samples.Among common buckwheat samples we observed best antioxidant properties for cultivars Bamby and KASHO-2, that was about one third lower than for tartary buckwheat.These two cultivars also contained relatively high amount of flavonoids.
Available literature documents, that tartary buckwheat because of higher polyphenol and flavonoid content exhibit higher antioxidant properties (

SERINE PROTEASE INHIBITION
Data from the literature indicate potential inhibitory activity of buckwheat extracts to set of enzymes including serine proteases.This inhibitory activity authors ascribe mainly to various peptides present in buckwheat seed (Tsybina et al., 2004).
Because flavonoids are known for their inhibitory action to various enzymes (Jedinák et al., 2006), we decided to test buckwheat hull samples with proven flavonoid content for inhibition of serine proteases trypsin, thrombin, urokinase and elastase.Results were expressed in IC 50 values, that represent extract concentration with 50% inhibitory activity in comparison with control (without an inhibitor).Determinations are listed in Table 3.As we can see, authors investigated protease inhibitory activity of buckwheat mainly to trypsin, chymotrypsin, subtilisin and cathepsin G.To our knowledge this is for the first time that was examined buckwheat extract inhibition of thrombin, urokinase and elastase.It seems that flavonoids are in this case effective components from buckwheat hull extracts.

CONCLUSION
Buckwheat belongs to traditional crops in Central and Eastern Europe and Asia.It is effective in management of many diseases, mainly cardiovascular and digestion disorders, cancer, diabetes and obesity.Effective prophylactic compounds are present mainly in outer layers of buckwheat grain.
In this study there were screened hulls of ten buckwheat cultivars.We can conclude, that the highest total flavonoid content revealed tartary buckwheat Madawaska.Among common buckwheat best values achieved cultivars Bamby and KASHO-2.Samples with highest flavonoid content were the most effective in testing of their antioxidant and antiproteinase properties.In regard of achieved results we can commend tartary buckwheat Madawaska and common buckwheat cultivars Bamby and KASHO-2 for further experiments.

Table 1 .
Overview of tested buckwheat cultivars.

Sedej et al., 2008; Markovic et al., 2009).
But there were some that claimed no relationship in this case (Oomah, Mazza, 1996).Our results show no significant

Table 3 .
Inhibitory effects of buckwheat hull extracts to serine proteases trypsin, thrombin, urokinase and elastase.