EVALUATION OF TOTAL POLYPHENOL CONTENT AND ANTIOXIDANT CAPACITY OF DIFFERENT VERITY LUPIN SEEDS

Legumes, including lupins, beans, lentil and chickpea, are one of the most important crops in the world because of their nutritional quality. Lupin seeds have been used as human food and animal feed since ancient times. It was known that antioxidant photochemical in foods have many health benefits including prevention of various diseases associated with oxidative stress such as cancer, cardiovascular disease, neuro-degeneration and diabetes. Lupin grains are rich sources of complex carbohydrates, protein, vitamins and minerals. Antioxidants can be found naturally in foods. Total polyphenols content and antioxidant activity were measured in four varieties of lupin, namely in white lupin, blue lupin, yellow lupin and Mutabilis lupin species. A majority of antioxidants naturally present in foods occur in phenolic structures and especially in flavonoid structures. The content of the total polyphenols was determined by using the Folin-Ciocalteu reagent (FCR). Antioxidant activity was measured by using a compound DPPH ̇ (2.2-diphenyl-1-picrylhydrazyl). In the present experiment according to the average contents of total polyphenols (TPC) in dry matter of lupin seeds there was the following line: L. Angustifolius (blue) lupin (696.212 mg GAE.100g-1) > L. Albus (white) lupin (614.13 mg GAE.100g-1) > L. Luteus (yellow) lupin (467.78 mg GAE.100g-1) > L. Mutabilis (pearl) lupin (367.36 mg GAE.100g-1). Based on the measured values of total antioxidant capacity (TAC) of lupin samples can be classified as follows: L. Albus (white) lupin (43.44%) >L. Angustifolius (blue) lupin (38.27%) >L. Luteus (yellow) lupin (22.29%) >L. Mutabilis (Pearl) lupin (20.80%). The relationship of antioxidant capacity with total polyphenolic was discussed. According to used statistical analyzes. Correlation between the phenolic contents and antioxidant capacity was significantly positive (r = 0.88). Our results confirmed that legumes can be a good source of bioactive compounds in the human nutrition. The main objective of the present work was to evaluated the content of total polyphenols and an antioxidant capacity of four Lupine species.


INTRODUCTION
Legumes used by humans are commonly called food legumes or grain legumes.The food legumes can be divided into two groups, the pulses and the oilseeds.Grain legumes can offer ecosystem services such as renewable N input, the improvement of soil fertility and the diversification of cropping systems, Jensen et al. (2012).Animal proteins being more expensive, especially people in developing countries depend largely on plant to fulfill their protein requirements.Moreover, it is also a good source of minerals (Kirmizi and Guleryuz, 2007).Leguminous seeds present one of the most promising alternative protein sources for the nutritional supplementation and technological improvement of traditional foods (Martinez-Villaluenga et al., 2009).Lupin is a representative of legume family which includes over 450 species are known, from which only four are of agronomic interest Reinhard et al. (2006).L. Albus L.: white lupin, L. Angustifolius L.: blue or narrow-leafed lupin, L. Luteus L.: yellow lupin and L. Mutabilis L.: pearl or Tarrwi lupin (Reinhard et al., 2006;Uzun et al., 2007).The first three species originate from the Mediterranean area, including Turkey, while L. Mutabilis belongs to South America (Mulayim et al., 2002).Lupinus Albus native to Mediterranean area is agriculturally important (Kirmizi and Guleryuz, 2007).white lupine and spring wheat or spring triticale, was most successful in yield and protein content.Also, harvest dates are as crucial as seeding rates for lupin/cereal forage because time of harvest determines the stage of maturity and therefore, forage quality.Harvesting between 116 -130 days is recommended (Azo et al., 2012).Lupin seeds have been used as human food and animal feed since ancient times.White lupine can be a phosphorus efficient plant and could help reduce the need for P fertilizer and enhance yields.It forms cluster roots in response to phosphorus starvation (Cheng et al., 2011).The bitter seeds contain the quinolizidine alkaloids lupanine and

MATERIAL AND METHODOLOGY
Material: Lupin Samples were collected from the market, but information such as the variety, were not mentioned, four species of lupin seeds (L.Albus (white), L. Angustifolius (blue), L. Luteus (yellow), L. Mutabilis (pearl) were purchased.
Extraction: For 12 hours extraction, dry material (5 g) was used and continuously extracted by a Twisselmann extractor with methanol (80%, v/v).

Determination of total polyphenols
Total polyphenols were determined by the method of Lachman et al. (2003) and expressed as mg of gallic acid equivalent per100 g dry mater.Gallic acid is usually used as a standard unit for phenolics content determination because a wide spectrum of phenolic compounds.The total polyphenol content was estimated using Folin-Ciocalteau assay, Folin-Ciocalteu reagent was added to the extract solutions.After 4 min, 5 mL of Na2CO3, (20%) was added, the volume was adjusted to 50 mL by adding distilled water and the mixture was stored at room temperature for 2 h, the absorbance values was measured at 765 nm of wavelength against methanol blank on the spectrophotometer Shimadzu 710 (Japan).The concentration of polyphenols was calculated from a standard curve plotted with known concentration of gallic acid.
2.2-diphenyl-1-pikrylhydrazyl (DPPH˙) was pipetted to cuvette (3.9 cm³) then the value of absorbance, which corresponded to the initial concentration of DPPH˙ solution in time Ao was written.Then 0.1 cm of the lupin extracted was added.Solution in the cuvette was mixed and then was immediately started to measure the dependence A = f (t).The absorbance after10 minutes was measured at 515 nm in the spectrophotometer Shimadzu UV/VIS-1240 was mixed and measured.The percentage of inhibition reflects how antioxidant compound are able to remove DPPH˙ radical at the given time.

Statistical analysis
Results were statistically evaluated by the Analysis of Variance.All the assays were carried out in triplicates and results are expressed as mean ±SD.The data were subjected to the F-test in the one-way analysis of variance (ANOVA) If the p-value of the F-test is less than 0.05, there is a statistically significant difference between the means at the 95% confidence level; the Multiple Range Tests will tell which means are significantly different from which others.The method currently being used to discriminate among the means of Fisher's least significant difference (LSD) procedure.Analysis was conducted using SAS software 9.4.

RESULTS AND DISCUSSION
On the base of reached results there were estimated changes in the total polyphenols content and also changes in total antioxidant capacity values in dependence on varieties of lupin seeds.

Statistical evaluation of antioxidant capacity(TAC) differences significance within the frame of chosen species
Another indicator that has been evaluated and compared was the changes in total antioxidant capacity values in lupin seeds.This method is based on decreased absorbance of DPPH radical read at 515 nm, due to the action of antioxidants.A 0.1 mL volume of the sample was added to 3.9 mL of DPPH• dissolved in methanolic solution and carefully homogenized.Free radical was freshly prepared before each assay while protected from light.The DPPH method is a preferred method because it is fast, easy and reliable and does not require a special reaction and device.The free radical scavenging activities of extracts depend on the ability of antioxidant compounds to lose hydrogen and the structural conformation of these components (Shimada et al., 1992).The DPPH free radical, which is at its maximum wavelength at 517 nm, can easily receive an electron or hydrogen from antioxidant molecules to become

DPPH• +A-H→DPPH-H + A•
In the present work as shon in (Table 1, 2; Figure 1, 3), it was found that, antioxidant capacity in samples ranges from 20.795% to 43.4363%.Statistically the highest value of antioxidant capacity (43.4363%) was recorded in L. Albus species (white color).The lowest value of antioxidant capacity (20.795%) was recorded in L. Mutabilis species (pearl color).In the case of L. Angustifolius (blue color) was (38.2685%), and (22.2919%) for Luteus species (yellow color).Ahmed (2014) published that the value of antioxidant capacity in lupine flour was (20.26 ±1.22).Gamal and Ismail (2013) determined that antioxidant activity by DPPH (Inhibition %) was (19.21 ±0.345%) in lupin seeds, (Lupinus termis L.).All the assays were carried out in triplicates and results are expressed as mean ±SD.The data were subjected to one-way analysis of variance (ANOVA) and the differences between various concentrations were determined Fisher LSD test using SAS software., and in L. Angostifolius unknown variety name was (0.424 mg Trolox Eq.g -1 ), in two varieties of L. Albus were (0.153 mg Trolox Eq.g -1 ) In comparison to our measured values of antioxidant activity their result was similar to the present value in L. Luteus with (22.29%) and present result in L. Albus with (43.44%) higher than their results in L. Albus but present result in L. Angostifolius with (38.27%) lower than their result in L. Angostifolius with (0.424 mg Trolox Eq.g -1 ).

Correlation between the total antioxidant activity values and total phenolics contents
ANOVA linear correlation coefficients were used to assess the relationships between TPC and TAC, Correlation: Our result confirmed a strong statically correlations between total polyphenol content and total antioxidant capacity values.A statistically strongly significant correlation (R = 0.88; p <0.05) was found (Figure 4).Wang and Clements (2008) found that the free radical scavenging activity is not well correlated with total phenolic contents (R = 0.204) in different species and varieties of lupin seeds.Amarowicz et al. (2005) analyzed the extracts of fababean, broad bean, adzuki bean, red bean, pea, red lentil and green lentil seeds using 80% (v/v) acetone and confirmed a statistically significant correlation between the total antioxidant activity values and total phenolics (p = 0.01).A strong correlation between total polyphenol content and antioxidant activity (R = 0.86; p <0.05) was observed also by Akond et al. (2011) in common bean and a statistically strongly significant correlation (p-value 2.391E-06; R = 0.802) was found between total ±8.35 μg GAE.g -1 d.w) which equal to 1381.7 ±8.35 mg CAE.100g -1 )].Also Walaa et al. (2015)

Figure 1
Figure 1Average content of total polyphenols TPC (mg GAE.100g -1 ) and Average content of total antioxidant capacity TAC (%) in chosen four species of lupin seeds.
a stable diamagnetic molecule (Soares et al., 1997).Owing to the DPPH radical's ability to bind H, it is considered to have a radical scavenging property.A solution of DPPH radicals prepared in methanol is converted into DPPH-H (diphenylhydrazine) molecules in the presence of an antioxidant agent, as shown in the following equation.Discoloration occurs due to the decreasing quantity of DPPH radicals in the environment.The discoloration of the DPPH therefore reflects the radical scavenging activity of the analyzed extract (Guo et al., 2007).The method is based on the reduction of alcoholic DPPH• solutions in the presence of a hydrogen donating antioxidant (AH) to the non radical form DPPH-H.

Figure 2
Figure 2Box-plot of the content of total polyphenols TPC (mg GAE.100g -1 ) in four species of lupin.L. Albus L. Angustifolius L. Luteus L. Mutabilis

Figure 3
Figure 3 Box-plot of the content of total antioxidant capacity TAC (%) in four species of lupin.

Figure 4
Figure 4Correlation between TP and TAC of four species of lupin.

Zielinska et al. 2008; Martinez-Villalunga et al. 2009). White lupine seeds are generally classified as sweet or bitter depending on the alkaloid content, which ranges from 0.01 to 4% (Bhardwaj and Hamama, 2012). It is an interesting grain legume due to its low alkaloid lines and higher protein content (34 -45 %), similar to that of soybean (Annicchiarico, 2008; Laudadio and Tufarelli, 2011). Lupine
(Sebastia et al., 2001)u et al., 2009)resent in foods occur in phenolic structures and especially in flavonoid structures.In addition to fiber, legume grains also contain many substances to improve health such as vitamins, minerals, and other substances, including phenolic compounds (Afshin et al., 2014).Phenolic compounds are resistant to oxidation and protect cell damage to prevent the risk of degenerative diseases thanks to antioxidative, anti-inflammatory, antiallergic and anticarcinogenic activities.(Nderituetal., 2014;Xu et al., 2009).Cholesterol-free legumes in combination with their low sodium content form a good food stuff not only for people living in developing countries but also for those living in industrialized nations(Sebastia et al., 2001).

Martínez-Villaluenga et al., 2006). However
, although lupin seed is one of the legumes with the lowest levels of non-nutritional compounds (trypsin inhibitors, phytic acid, saponins and lectins)

by Martinez- Villaluenga et al
Lupins have traditionally been used for animal feed but are gaining recognition as a health food for humans, due to their unique dietary composition.Total phenolics and antioxidant activities of grains and germinated grains have been widely investigated.Germination produced high phenolic content and consequently increased the antioxidant activities of lupine seeds.Legumes have been shown to help manage both In general, the human body has its own natural antioxidant system to stand against free radicals using certain enzymes.Antioxidants can be found naturally in foods (Kedare and Singh, 2011).A majority of antioxidants naturally present in foods occur in phenolic structures and especially in flavonoid structures.Sweet lupins, such as those grown in . (2008).In contrast to other leguminous plants such as peas, soy beans, Lupinus Albus contain extremely low amounts of trypsin inhibitors, lectins, isoflavones, saponins and cyanogens (Joray et al., 2007, Zralý et al., 2007).cholesterol and blood glucose (Bazzano et al., 2011: Sievenpiper et al., 2009).Increased intakes are linked to the prevention of heart disease, diabetes and some cancers (Bazzano et al., 2001; Hwang et al., 2009).It is found that phytochemicals from legumes may be responsible effects (Campost-Vega et al., 2010).The interest in natural antioxidants has increased considerably in recent years because of their beneficial effects of prevention and risk reduction in several diseases.Due to low glycemic index of lupin seeds, it was found that lupin kernel fibres have appetite suppression (Archer et al., 2004) and cholesterol lowering properties, that they lower blood glucose and insulin levels (Hall et al., 2005).The profiles and quantities of polyphenols and tannins in legumes are affected by processing due to their highly reactive tances, which are considered to be natural antioxidants (Rybarczyk and Amarowicz, 2007; Dueñas et al., 2009) nature, which may affect their anti-oxidant activity and the nutritional value of foods (Dlamini et al., 2009).The quantitative determination of phenolic compound content as well as their antioxidant capacity may provide valuable information in considering health-promoting properties of lupin seeds.Lupin does not contain gluten, thus it is sometimes used as a functional ingredient in gluten free foods (Scarafoni et al., 2009).There is potential risk of lupine allergy appear (Zielinska et al., 2008).diabetes (American Diabetes Association, 2008).The phenolic content and composition of L. Angustifolius, despite its weak antioxidant capacity, may have positive implications for reducing the risk of cardiovascular disease due to its protective effects on blood vessel health (Oomah et al., 2006).Moreover, lupin seeds also contain constituents that alter satiety and other features of human health with the prospect of pharmaceutical potential (Lee et al., 2006; Duranti et al., 2008).

Table 1
Total phenolic content and antioxidant capacity (mean and standard deviation values).In chosen species of lupin.Data expressed as means of six replications ±standard deviation.Values in the same column with different letters present significant differences p <0.05 using F-test for independent samples.

Table 2
Total phenolic content and antioxidant capacity (mean and standard deviation values with the group).In chosen four species of lupin seeds.