Phytochemical characterization, evaluation of protein content and bioprospection of lectins and trypsin inhibitors in extract of Sesbania virgata (Cav.) Poir Seeds

Fabaceae presents 145 subclades and 737 genera. Among them, the genus Sesbania, with about of 500 species, such as Sesbania virgata, whose seeds present high protein content. The present study aimed to characterize phytochemically, to evaluate the protein content and to bioprospect lectins and trypsin inhibitors in the protein extract of Sesbania virgata seeds. Seed meal was used to obtain Total Protein Extract (TPE) in Glycine-NaOH 0.1 mol.L pH 9.0 NaCl 0.15 mol.L (1:15, w/v). After 22 hours of magnetic stirring, the extract was centrifuged (7.076.3 x g, 30 min, and 4 °C) and the supernatant collected and named TPE. In this, it was determined the total protein content using the bovine serum albumin standard curve; bioprospecting lectins by hemagglutination assays in rabbit erythrocytes (CEUA/UFPB n° 178/2015), and trypsin inhibitors by enzyme assays using the enzyme bovine trypsin; and phytochemical characterization was given by different qualitative tests. TPE presented as protein content 7.13 mgP/mL and 106.96 mgP/gF; hemagglutination titers were 32 UH/mL, with 480 UH/gFa and 4.4887 UH/mgP; not precipitated trypsin inhibitors; and in phytochemical tests, reacted positively to saponins, catechins, flavones, flavonols, xanthones, anthocyanins, anthocyanidins SciForum and flavonoids, but reacted negatively to pyrogallic and catechin tannins, leucoantocyanidins, steroids and triterpenes. In other assays performed by our group (data not shown) in which an extract was obtained from buffer Glycine-HCl 0.1 mol.L pH 2.6 NaCl 0.15 mol.L, under the same conditions, it was found that this extraction method was not able of bioprospecting lectins, but favored the precipitation of protease inhibitors. It is understood, thus, that the extraction method influences the obtaining of TPE, the demarcation of considerable protein content and numerous phytochemical compounds, besides the hemagglutination assays suggest the presence of lectins. Together, all these constituents make TPE a versatile biotechnological tool, to check various biological activities.

Commonly, legume seeds exhibit biological activities by the mechanism of action of proteins, as the lectins, an antinutritional factor of protein origin with ability to agglutinate erythrocytes and/or to precipitate glycoconjugates (Rüdiger, 1998), mediated by the structure or similarity between the binding sites present on the surface of their membranes (Santana et al., 2008). As well as, protease inhibitors, protein factors with skillful ability to inhibit the catalytic activity of numerous proteolytic enzymes (Neurath, 1989;Ryan, 1990;Brzin & Kidric, 1995).
The properties displayed by its legume seeds encourage the development of natural or synthetic bioactive products capable of being employed as biotechnological tools. Thus, the present study aimed to characterize phytochemically, to evaluate the protein content and to bioprospect lectins and trypsin inhibitors in the protein extract of Sesbania virgata seeds.
Thus, it is possible to infer that the biological activities and bioprospection of biologically active proteins, as they occur without the interference of tannins, in particular, will be free of the possibility of false-positive results, as commonly occurs in lectin activity tests.
Leucoantocianidins, steroids and triterpenes also were not detected ( Table 1), suggesting that their non-precipitation is a result of insoluble complexes that form between proteins or carbohydrates present in TPE, as well as by the action of the solvents used.

Soluble protein content and bioprospection of lectin and trypsin inhibitors
The soluble protein content present in TPE was 7.13 mgP/mL and 106.96 mgP/mgF. Hemagglutination titers were 32 UH/mL, with 480 UH/gFa and 4.4887 UH/mgP and the lectin activity is seen with 60 minutes of starting the test and remaining for up to 24 hours after. TPE do not have apparent antitryptic activity.
In other assays performed by our group (data not shown) in which an extract was obtained from buffer Glycine-HCl 0.1 mol.L -1 pH 2.6 NaCl 0.15 mol.L -1 , under the same conditions, it was found that this extraction method was not able of bioprospecting lectins, but favored the precipitation of protease inhibitors.
Mol2Net, 2015, 1(Section A, B, C, etc.), 1-x, type of paper, doi: xxx-xxxx 3 This buffer showed values ranging from 62.99% to 74.16% inhibition, and 0.71 and 6.96 mg of inhibited trypsin/g sample, proving that for Sesbania virgata seeds, it is necessary that this protease inhibitor be extracted in alkaline pH.
While the extract obtained of the Glycine-NaOH (0.1 mol.L -1 , pH 9.0) containing NaCl 0.15 mol.L -1 , was chosen as the most effective for to bioprospect lectins. Colorimetric -

Phytochemical partial characterization
The phytochemical partial characterization was determined by colorimetric reactions, according to methodologies prescribed by Barbosa et al. (2001), Matos (1997) and Robbers et al. (1997).

Determination of soluble protein content
The soluble protein content was determined by colorimetric reactions, using bovine serum albumin as standard and Coomassie Brilliant Blue G-250 as chromogenic, according Bradford (1976).

Lectin and trypsin inhibitors detection assays
Detection of lectin activity was performed using haemagglutination assays against 3% rabbit erythrocytes (Debray et al., 1981). The entire procedure was approved by the Ethics Committee for the Use of Animals (CEUA/UFPB, nº 178/2015).
Antitryptic activity detection assays, in the presence or absence of trypsin inhibitors, were performed according by Xavier-Filho et al. (1989), using bovine trypsin and DL-BAρNA as its chromogenic substrate.

Conclusions
It is understood, thus, that the extraction method influences the obtaining of TPE, the demarcation of considerable protein content and numerous phytochemical compounds, besides the hemagglutination assays suggest the presence of lectins. Together, all these constituents make TPE a versatile biotechnological tool, to check various biological activities.