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Food security is major issue as more food will be required due to population growth, climate change and water shortage. There has been an increasing demand worldwide for food proteins either from animal or vegetable sources. Data from the Food and Agricultural Organization (FAO) shows that total protein production in the world in 1980 reached 290 million tons, including 79% from vegetable sources against 21% from animal origin. Oilseeds are the second major source of protein production (16.9%) after cereals (53.7%).
The animal source of protein is expensive considering the market price, environmental impact and the loss of vegetable protein and energy, under industrial conditions, when converting into animal protein. On the other hand, cereals are an excellent source of proteins, followed by oilseeds which have great potential as seen by soybeans, and to a small extent rapeseed, pea, cottonseed, ground nut, linseed and sunflower.1,, &
Sunflower seeds are particularly interesting in view of their widespread availability in areas where soy is not or only produced on a small scale. Sunflower seed meal (SFM), which is a by-product from oil extraction has great potential as a protein source for human consumption, but unfortunately is not well explored due to unsuitable oil extraction methods that can serve both purposes, namely (1) optimum oil extraction and (2) without denaturing or affecting the physicochemical properties of proteins; For SFM the cost/benefit remains unattractive for the manufacturers, therefore a great part of SFM is intended for animal feed production.
Furthermore, proteins from SFM can be isolated from oilseeds and used to prepare value-added functional proteins and peptides not only for food applications as an emulsifier 2, fortification of yogurts, meat and milk products, infant formulae, bakery products and pasta products,but also for specific biological activities including the inhibition of angiotensin-1- converting enzyme (ACE) which regulates blood pressure and electrolytes, that will be discussed later in this chapter.
The aim of this research is to:
Isolate sunflower protein,
Hydrolyse the proteins with digestive enzymes
Separate the hydrolysates into different molecular weight (MW) fraction and
Assess its potential antioxidant activity and ACE inhibitory activity.
Amino acids, peptides and protein
Amino acids (AA) are the building blocks of every protein, linked one to another by a covalent peptidebond. There is a set of 20 amino acids that is fundamental for human life, and when arranged together these amino acids can yield a variety of product such as hormones, antibodies transporters, enzymes, muscle fibers, poisons, and several others substances with specific biological activities.
Structure of an amino acid
All amino acids share two common structures, a carboxyl group and an amino group bonded to the same carbon denominated alpha-carbon. Another structure is the side chain, or R group, that give an amino acid its chemical properties. Furthermore, AA can be grouped in 5 classes based on their chemical properties as shown in figure 1, more specifically on their predisposition to interact with water at a biological pH (approximately 7.0), in other words its polarity.
Non-polar aliphatic R groups
Amino acids glycine, alanine, proline, valine, leucine, isoleucine and methionine are nonpolar and hydrophobic. Glycine with the small R group does not contribute to hydrophobic interactions. Methionine and cysteine are two sulphur containing amino acids, which have important characteristics when interacting with other sulphur containing amino acids, creating a disulphide bond that helps to stabilize structure in tertiary protein. Alanine, valine, leucine and isoleucine have a tendency to aggregate within proteins, by hydrophobic interactions in order to stabilize proteins structures. Proline, is considered an imino acid, its secondary amino structure forms a cyclic structure in its side chain, which reduces flexibility in polypeptides structures.
Aromatic R groups
Composed of phenylalanine, tyrosine, and tryptophan, this group has a characteristic aromatic ring in its side chain structure. They are less hydrophobic than non-polar aliphatic groups, particularly tyrosine and tryptophan, the first due to its hydroxyl group and the second due to nitrogen in its indole ring.
Figure EXTRACTED FROM LEHNINGER PRINCIPLES OF BIOCHEMISTRY, FOURTH EDITION - DAVID L. NELSON, MICHAEL M. COX, PAG 79.
Polar uncharged R groups
The amino acids of this group are serine threonine, cysteine, asparagine, and glutamine. Amino acids of this group are more hydrophilic than the non-polar group, due to their functional groups in the side chain structure; they can interact with water by hydrogen bonds, and therefore have increased solubility in water. Moreover, serine and threonine have a hydroxyl group, asparagine and glutamine an amide group is part of the side chain. Cysteine contains sulphur and when oxidised and linked by disulfide bond it forms a dimeric amino acid called Cystine, but alone can form hydrogen bonds with water.
Positively charged (basic) R groups
These amino acids are highly in soluble in water; at pH 7.0 the side chain has considerable positive charges. Lysine, arginine and histidine are part of this group, lysine has two amino groups, at the Îµ position on its aliphatic chain and the other on its Î±-carbon; arginine has a functional guanidine group attached to its side chain and histidine has an aromatic group imidazole.
Negatively charged (Acidic) R groups
This group is highly soluble in water, hydrophilic, with negative charges near pH 7.0 due to its secondary carboxyl group. The amino acids related to this group are aspartate and glutamate. They are the hydrolysed form of, respectively, asparagine and glutamine in an acid or base.
Molecular forces governing protein interactions
It is important to understand the molecular forces behind the structure of proteins and their influence on their functional properties particularly when exposed in different aqueous environment (polar or non-polar). Moreover, there are intramolecular and intermolecular forces the first regard to the attraction forces that exist between atoms within a molecule, the late to the attraction between stables molecules in a compound. Moreover, Intermolecular forces are based on electrostatic interactions, they happen over great distances, and its intensity vary inversely to distance, also depends on the electrical charge of protein molecules. The force may be repulsive for like charge and attractive for opposite charges. The types of molecular interactions are: Hydrogen bonds, dipole - dipole bonds and London dispersion forces and hydrophobic interactions.
Hydrogen Bonds are an electrostatic interaction force between polar molecules where two atoms with partial negative charges share with a partial positive charged hydrogen. This interaction happens normally between acid N-H and base C=O groups in either alpha helices or beta sheets.
Dipole - Dipole electrostatic interactions are formed when there is a large difference in electronegativity between two atoms bonded together in a covalent bond. This causes the shared pair of electrons to be shared unequally. They are pulled towards the more electronegative atom.
The London dispersion force or Van der Waals forces are a temporary weak attraction between two atoms due to an electron cloud that exist around the proton of an atom, there is an ideal distance so Van der Waals forces take place
Hydrophobic interactions exist when a non-polar substance is added in polar aqueous environment, with the absence of hydrogen bonding there are not either attracting or repelling forces between both substances. A classical example is the fact that oil don't mix in water.
Furthermore, covalent bound is the strongest type of intramolecular force, it takes place when two atoms shares a pair of electron, with a heat formation of 100kcal/mol covalently bonded materials generally have very high melting points and are generally insoluble. There is also the covalent disulphide bond not as strong as covalent with a heat formation of 50kcal/mol, the link between two cysteine residues depends on the conformation of the peptide chain.
Peptides and Protein
Peptides and proteins are polymers of amino acids linked by peptide bonds. Peptide bonds are formed by a condensation reaction (see figure 2), an Î±-carboxyl group of one amino acid is linked to Î±-amide group of another amino acid with a formation of a water molecule as a by-product of this reaction. Two amino acids linked together are called dipeptide and more than two are called oligopeptides and even polypeptides, the difference being in the molecular weight, polypeptides have a molecular weight below 10,000 in comparison to proteins.
Structure of Protein
Protein may have three-dimensional structures; this arrangement in a protein is known as conformation. When hydrolyzed the product is a linear polymer mix of amino acids not necessarily in proportion, and may be a combination of three, eight or more amino acids.
A simple linear sequence is called the primary structure formed by covalent bonds. The secondary structure may have a characteristic conformation due to interactions in different regions along the peptide, first the Î±-helix conformation, named because the structure of the polypeptide is round up around an imaginary axis drawn longitudinally through the middle of the helix. The structure is stabilized by intra-chain hydrogen bonds and dipole interactions. The second conformation is the Î² sheets, where the backbone of the polypeptide is extended into a zigzag. The Î² turns, works more like a connecting function, linking Î± helix or Î² sheets conformations.
Furthermore, the tertiary structure is basically the folding of polypeptides in a three-dimensional conformation forming a subunit, this structures may be stabilized by disulphide bonds if at least two molecules of methionine and/or cysteine are present in the polymer. Quaternary structure relates to a protein with at least 2 subunits.
Figure Figure extracted from Lehninger Principles of Biochemistry, Fourth Edition - David L. Nelson, Michael M. Cox, pag 85.
Caracterization of Sunflower Proteins
The protein of sunflower (Helianthus annuus) seed flour contains: Helianthinin or 11S globulin (38.33%), albumins 2S (39.04%), glutelin (17.09%) and prolamin (05.54%). Regarding the structure of helianthinin is known that it is an oligomeric protein with a molecular weight (MW) between 300-350 kDa. Helianthinin is mainly 11S (hexamer), but depending on the pH and temperature it can dissociates to 7S (trimer), and 2-3S (monomeric).
There are evidences that each subunit of 11S globulins possess an acidic (32-44 kDa) and a basic (21-27 kDa) polypeptide linked by a single disulphide bond the same occur with other seed proteins such peas, soy or lupin.
Regarding to nutritional content, Sunflower protein have no anti-nutritional components such as toxins or proteases inhibitors and its amino acids profile composition complies with the Food and Agriculture Organization (FAO)1, however
It contains 3 to 5% of polyphenols which accounts for as a limiting component that avoid the large scale use in food for human consumption, and also it is important outline that the unsuitable method of oil extraction as in the preliminary preparation for example: drying at high temperatures, dehulling and conditioning or desolventizing-toasting in oil extraction surely deteriorates the protein reducing either Its nutritional and physicochemical properties such as solubility, emulsion and foaming capabilities.
Figure COVALENT BONDING OF PHENOLIC ACID TO PROTEIN.
Interactions between phenolic compounds and sunflowoer seeds proteins
The major phenolic compound is chlorogenic acid (3-0-caffeoyl - D - quinic acid), however interactions with basic, acidic or enzymatic hydrolysis brakes the ester linkage and give caffeic acid and D - quinic acid as a products, hence the presence of those 3 phenolic compounds in processed sunflower products.
As in relation to enzymatic hydrolysis, polyphenols oxidase, an enzyme responsible to oxidize 70% of total chlorogenic acid, is naturally present in sunflower seeds. Furthermore, there evidences that caffeic acid and D - quinic acid are responsible for the irreversible covalent bonding with S as show in figure 3 and N groups of sunflower protein at higher pH or if at low pH by hydrogen bonding, ionic, and Hydrophobic intereactions.
Proteins may be precursors of bioactive peptide products after enzymatic digestion in the gastrointestinal tract or during industrial food processing, they are of great interest for food and supplement manufactures and pharmaceutical industry as constituents of functional foods or as a nutraceutical products. Furthermore, Nutraceutical is a term used to define a range of processed foods, dietary supplement, herbal product or biologically active compound that prevent or treat some types of health condition e.g. hypertension. On the other hand functional foods are foods that contain natural ingredients such fibers, polyunsaturated fatty acids, minerals and oligosaccharides that have been proved to benefit health or well being and reduce the risk of disease in addition to supplying the standard nutrients.
Therefore the sunflower protein hydrolysate may be a source of bioactive peptides that plays a role either by decreasing blood pressure, regulation of cardiovascular, nervous immune system, antioxidative, antimicrobial, and probably other functions not yet discovered my food scientists.
As the knowledge of healthy benefits of antioxidants are wide spread nowadays, started to become of great interest for food and supplement manufacturers as there are several applications for antioxidants either in food systems as lipid oxidation, retarding discoloration therefore extending shelf life and for health related issues.
Free radicals may be defined as chemical species that has an odd or unpaired electron. They are neutral, short lived, unstable and highly reactive to pair up the odd electron to finally achieve stable configuration. Moreover, the two major types of free radicals can be derivative from (1) reactive oxygen species (ROS) and (2) Reactive nitrogen species (RNS), Types of ROS include the hydroxyl radical, the super oxide anion radical, hydrogen peroxide, singlet oxygen, hypochlorite radical, and various lipid peroxides, as for RNS depending on the environment nitric oxide (NO) can be converted to nitrosonium cation (NO+), nitroxyl anion (NO) or peroxynitrite (ONOO).
To avoid the cells and organ damage of the body from the action of ROS and RNS substances, humans possess endogenous mechanisms that functions interactively and synergistically to neutralize free radicals and antioxidants from nutrition source also help stabilizing free radicals before they attack cells. Antioxidants are absolutely critical for maintaining optimal cellular and systemic health and well-being as well as food integrity for long period.
Moreover, naturally there is a dynamic balance between the amount of free radicals produced in the body and antioxidants to scavenge or quench them to protect the body against deleterious effects. An oxidative stress may result if antioxidants fall under normal physiological level as the body decrease the ability to get rid of these radicals from the metabolism. Consequences are damage of cell membrane, nucleic acids, proteins and enzymes and other small molecules, resulting in cellular damage and loss of function, that contribute to aging and degenerative diseases of aging such as cancer, cardiovascular disease, immune system decline, diabetes mellitus, inflammation, renal failure and others.
There are several methods used nowadays to determine antioxidant activity of food extracts and beverages including 1,1-diphenyl2-picrydrazyl DPPH radical scavenging assay, Trolox Equivalent Antioxidant Capacity (TEAC), Total Radical - Trapping Antioxidant Parameter (TRAP), The Ferric Reducing Ability of Plasma (FRAP), Oxygen Radical Absorbance capacity method (ORAC). Hydroperoxides (PV) and aldehydes (TBARS) were the methods of choice for this project.
ACE Inhibitory Activity
The precursor of the ACE inhibitors was discovered in 1948 by Mauricio Rocha e Silva and was called a bradykinin noted by its potent, though very short, action as an hypotensive and as smooth muscle stimulator extracted from the venom of Bothrops jararaca.,Due to its very short action the interest of the pharmaceutical industry was to create a substance to prolong the peptide activity, which later one was discovered the bradykinin potentiating factor, with increased duration and more powerful substance by Dr. Sérgio Henrique Ferreira.
Angiotensin-I-converting enzyme (ACE) is a ectoenzyme (peptidyldipeptide hydrolase, EC 184.108.40.206) associated in the Renin-angiotensin system (RAS) as shown in figure 4, that plays a role in the regulation of peripheral blood pressure, electrolyte homeostasis and cardiovascular function. Furtheremore, Ace is expressed in two different forms, somatic it is located mainly in the capillary beds of the lungs, but also present in other parts of the body, and germinal where it is expressed in the sperm. Its main function is to activate angiotensin II by cleaving two peptides from the decapeptide Angiotensin I to the octapeptide angiotensin II as shown in figure 3.
Figure SHOW the action of ACE-I cleaving the dipeptides from angiotensin I when converting to Angiotensin II
Angiotensin II is a multifunctional peptide responsible not only for the vasoconstriction but also acts on the outer cortex of the adrenal gland, via angiotensin II receptors, which in turn release aldosterone which increases the absorption of sodium and water in exchange for potassium which increases blood volume hence blood pressure. Another function is the inactivation of bradykinin, a peptide responsible for lowering the blood pressure in the kinin system as seen in figure 4.
Therefore ACE inhibitors are the main target in lowering hypertension which is a risk factor for heart disease and other related conditions. Several types of oral medicines from natural or synthetic sources have been used to treat congestive heart failure.
Figure ilustrate the role of ACE in the Renin - Angiotensin System AND KALLIKREIN-KININ SYSTEM.
ACE inhibitory peptides derived from food proteins
There are evidences showing that ACE inhibitory peptide can be found in food protein in different sources of food including, casein and whey protein from milk , soy bean, small red bean protein, rapeseed protein, kidney beans, sunflower protein9 and many others. Moreover, the procedure to identify and classify such peptides can be either from in vitro enzymatic, or in vivo gastrointestinal digests of the precursor protein and there is extraordinary situation where the ACE inhibitory peptides may be isolated without having to be hydrolysed for example garlic. Nevertheless, there are many evidences showing that ACE inhibitory peptides are made of 2-12 amino acids, therefore ultrafiltration technique has been used when achievement this particular size are required.
The effectiveness of the Ace inhibitory peptides is expressed as an IC50 value, where IC50 is the concentration required to inhibit 50% of the ACE activity. It is important to mention that food proteins may act as a substrate for ACE since the main activity of ACE is to cleave C-terminals dipeptides from non specific oligopeptides. Therefore, when preparing the samples in vitro there is a need to distinguish the substrates from true inhibitors, by preparing a sample with the peptides of interest and ACE together incubate for an hour before reading the ACE inhibitory activity is the method used.
The proteins may be separated in 3 groups, (1) the inhibitor type where the IC50 remains the same after incubation, (2) the pro - drug-type inhibitor where the inactive peptides are activated to true inhibitors either by ACE or digestive proteases and (3) the substrates per se with weak or no inhibitory activity at all.
Each food contains different proteins that after enzymatic digestion they generate different sequences of amino acid. In order to understand the mechanism of the reaction ondeti and Cushman (1982) made an analogy between ACE and Carboxypeptidase A, a zinc containing exopeptidase that cleaves a single amino acid from the carboxy-terminal end of peptides substrate.
An illustration of the carboxypeptidase A and ACE is shown in figure 5.
Figure Illustrate the structural subsite of carboxypeptidase A on the left and the Hypothetical structure of ACE suggested by ondeti and suchman (1982).
Figure Model suggested for different interactions between different groups of ACE inhibitors and the subsite S1, S'1 and S2 of ACE.
Based on the model presented in figure 6, structure-activity relationship studies from the venom peptide analog showed that the optimal carboxy terminal amino acid sequence that binds the enzyme was Phe-Ala-Pro, assuming that there is an interaction between the radical group of the respective amino acids and the subsites S1, S'1 and S'2 of ACE, and from there different classes of ACE inhibitors were developed with long duration and more potent with minimized side effects for example, Sulfhydryl-containing agents such as captopril, also Dicarboxylate-containing agents like enalapril, ramipril and others and finally Phosphonate-containing agent Fosinopril.
The positive side of the halotropics medicine cannot be contested. However they usually have side effects such persistent dry cough and drop in energy during treatment. Another solution would be an adaptation in the diet and lifestyle factors (such a decrease in weight, be more active physically, quitting smoke, and reducing stress) may have a significant impact.
Moreover, that's where functional foods play a role as a natural and safe that may prevent the onset of not only hypertension but other therapeutic purposes. Nevertheless there is a downside they are not as potent as the common drugs, and also most of the ACE inhibitory peptides with significant activity tested in vitro had not yet been tested in vivo there, therefore, there is a need to determine the efficacy of these peptides in vivo and to clarify the mechanism behind antihypertensive response of it and broaden the possible sources by continuing research such as in this work where aim and hope to find such peptides in sunflower seeds.
Material and Methods
The sunflower sample used was purchased from "Tesco brand". All reagents used were of analytical grade. Hexane, Methanol, ethanol, petroleum ether (grade 60-80), 3, 3, 3-Tetraethoxypropane (TEP), thiobarbituric acid (TBA) and glutaraldehyde standard, sodium thiosulphate, glacial acetic acid and chloroform were purchased from Sigma-Aldrich Company Ltd., Poole, England. All other products were obtained from Sigma-Aldrich. Before each practical procedure, a Control of substances hazardous to health regulation (COSHH 1988) Risk assessment form was read to each practical
Preparation of the Defatted Meal (DSM)
The dehulled seeds were milled in the laboratory grinder (MODEL OF GRINDER) until the meal became to a powder. Moreover, two methods were used for comparison hexane and Soxleht methods. For hexane extraction the meal was washed four times with hexane, meal solvent ratio used was 1:5(w/v), each wash during a period of two hours at 4°C and then at the last wash the sample was filtrated using Whatman N° 90 filter paper.
As for Soxleht method it was used a Soxtec semi automated apparatus ( MODEL?) using as a solvent petroleum ether 60-80), using the grounded seeds, 6 thimbles were weighed and numbered using a graphite pencil (1-6), then 6 grams of the dry sample was weighed and added in each thimble, also it was weighed the metal collection cup. All data was recorded in a piece of paper, relating to each thimble, its weight the weight of the sample and the weight of the metal collection cup.
The crude protein content from 3 different samples, (1) 2 samples defatted by hexane extraction, (2) 2 samples defatted using soxleht extraction and (3) 6 raw grounded sunflower samples. All were measured using the Kjeldahl method, (AOAC, 1984).Protein content factor used was N x 6.25.
The sample was wrapped in filter paper and placed in a Kjeldahl test tube. Then 2 Kjeldahl selenium tablets (catalyst) was added to the tube, also 20 ml of concentrated sulphuric acid to each tube. The tubes were placed in the digestion block, which was preheated to 450°C. The duration of Digestion was about 3 hours. Then the samples were cooled in the fume cupboard. The distillation of nitrogen from the digested sample is carried out in the Kjeltec 2200.
Sunflower Isolate Preparation (SIP)
During the protein isolation the main concern was the presence of phenol compounds chlorogenic acid; caffeic acid, and quinic acid as they react with protein especially low molecular weight (e.g. 2s fraction) forming irreversible covalent bonds that reduce organoleptic properties as well as solubility of proteins, also as a result of this interaction a dark colour and unpleasant smell from sunflower product, but also there is evidences of this interaction in rapeseed and soy plants
To overcome the problem five different methods of extraction were tested, the first two following the mild acidic extraction, the other three using alkaline extraction two of them with some modifications of the method. Follow below the description of each method used in detail.
Mild acidic extraction
Two different methods was extracted from the mild acidic protein extraction procedure established by Morr et al. (1985) with modifications by Pickardt C. et al.(2009), using 4 variables (1) Meal solvent ratio (MSR), (2) temperature (°C), (4) pH and NaCl (mol/L) an the values as specified in table 1
Using a dry sample of DSM (hexane) ratio (0.05: 1 W/V) in a NACl solution (2.98mol.L) in a beaker, then stirring (~200rpm) at temperature of 25°C, pH were adjusted to 6.0 and then adjusted every 15 minutes (0, 15, 30, 45, 60 minutes) using 0.1M HCL and NaOH to stabilize the pH as needed. Then, the beaker content was transferred to a container with a screw lid and putted through centrifuge at 20000g for 15 minutes at 20°C. The next step was to filter the supernatant using a whatman paper filter n°90.
For method 2 a ratio (W/V) 0.05 DSM hexane: 1 NaCl solution (2mol/L) in a beaker, then stirring (~200rpm) at temperature of 45°C, pH were adjusted to 7.4 and then adjusted every 15 minutes (0, 15, 30, 45, 60 minutes) using 0.1M HCL and NaOH to estabilize the pH as needed. Then, the beaker content was transferred to a container and closed with a screw lid and putted through centrifuge at 20000g for 15 minutes at 20°C. The next step was to filter the supernatant using a whatman paper filter n°90.
Alkaline extraction without sodium sulphite
A range of alkaline pH was tried in the protein extraction, the first attempt started with the mild acidic at ph 7.4 (see section 220.127.116.11 method 2). Furthermore, two procedures were carried on, at pH 10.5 and 12, and then using a ratio of 1:1 (W/V) 30 grams were dissolved in 300mls of deionized water and using a magnet stirrer, and a pH monitor (model) the pH was adjusted to 12 and left stirring for 1 hour. Then after 1 hour the solution was transferred to a container with a screw lid and taken to the centrifuge at 8000 x g for 15 minutes. The supernatant was separated and the isoelectric precipitation (section 7.2.9) procedure was used.
The same procedure was applied at pH 10.5 as described above.
Ethanol dephelolising prior to Alkaline extraction at pH 10.5
For this procedure using prior to alkaline extraction the sample was washed with ethanol 98% at a ratio of ??? and repeated this procedure until the dried sample did not show a yellow colour as a result of reaction with NaOH.
For alkaline extraction a ratio of 1:1 (W/V) 30 grams were dissolved in 300mls of deionized water, using a magnet stirrer and a pH monitor (model) the pH was adjusted to 10.5 and left stirring for 1 hour. Then after 1 hour the solution was transferred to a container with a screw lid and centrifuged at 8000 x g for 15 minutes. The supernatant was separated and the isoelectric precipitation (section 7.2.9) procedure was used.
Alkaline extraction at pH 10.5 with sodium sulphite
Using a ratio of 1:1 (W/V) 30 grams were dissolved in 300mls of deionized water, then 0.25% Na2SO3 for each 100 mL of deionised water was added, using a magnet stirrer and a pH monitor (model) the pH was adjusted to 10.5 and left stirring for 1 hour. Then after 1 hour the solution was transferred to a container with a screw lid and centrifuged at 8000 x g for 15 minutes. The supernatant was separated and the isoelectric precipitation (section 7.2.9) procedure was used.
This method was used to precipitate proteins from all extraction methods above, the supernatants pH was adjusted to the isoelectric point of sunflower meal protein (pH 4.3). Precipitates were recovered by centrifugation at 8000 X g for 20 minutes. Then washed with acidic water (pH 4.3) and centrifuged again at 8000 x g. Transferred to falcon tubes overed with nescofilm and stored in the freezer at -80°C overnight to be transferred next day for freeze drying. Holes was made in the nescofilm before transferring to the freeze dryer and it took about 1 week to become very dry.
Moreover, to prepare sunflower protein isolate (SPI) a mix pratio sample of (1:2.5w/v). 10gram was homogenized, by blender, in deionized water or 2 minutes. The pH was adjusted to 2.0 by 5N HCI. Pepsin (0.2857 gm) was added to the solution and the suspension was incubated at 37°C for 1 hour, stirring continuously using a magnet stirrer adjusting the pH as needed every 10 minutes. After one hour, the pH was adjusted to 5.3 by NaHC03 (0.9M) followed by the addition of 5N NaOH to a pH of 7.5. Then pancreatin (0.4 gm) was added and the mixture incubated at 37°C for a further 2 hours again under continuously stirring and pH checked every 10 minutes. After the completion of the digestion, the tube was submerged in boiling water in order to inactivate the enzyme and terminate the digestion. The digested peptides were then transferred to centrifuge tubes and centrifuged at 12000 X g for 15 minutes. The supernatant was then transferred to falcon tubes and covered with nescofilm and left in the freezer at -80°Cfor at least one night before transferred to freeze dryer. Holes was made in the nescofilm before transferring to the freeze dryer and it took about 1 week to become very dry.
Fractionation of protein
The steps taken for fractionation was Dissolving 1 gram of sample in 100 ml of deionized water, stirring for 10 minutes, then the solution was transferred to 10kDa cartridge( model) filter and centrifuged for 35 minutes at 3500 x g. after the upper layer was collected and labeled >10 kDa. The lower layer was placed in another cartridge filter pore size 5 kDa and centrifuged as above. Then the upper layer from this was labeled 5kDa<X< 10kDa. Repeating the same procedure but now using 2kDa cartridges for 35 minutes at 3500 x g, the upper layer was labeled 2kDa <x<5kDa and the lower layer x<2kDa. The supernatant was then transferred to falcon tubes and covered with nescofilm and left in the freezer at -80°Cfor at least one night before transferred to freeze dryer. Holes was made in the nescofilm before transferring to the freeze dryer and it took about 1 week to become very dry.
Measure the antioxidant activity
The antioxidant activity of the fractionated protein was measured using the method of Osawa et al. An emulsion was prepared. Moreover, a sample of 60mg was dissolved in 4.78ml of 50mM phosphate buffer (pH7), and then added to a linoleic acid solution of 0.18 ml and 10 ml of ethanol (absolute). Then the total volume was adjusted to 25ml with adjusted water. The solution was placed in a test tube covered with foil and closed with a rubber syringe plunger cap and sealed around it with nesco film and incubate in a dark room at 401°C. Then it was its oxidation rate was evaluated over 7 days using TBA and Peroxide value. All analyses were run in triplicate and averaged.
The peroxide value was measured according to the method of Mitsuda et al. . A 100Âµl sample from the reaction solution of the incubated linoleic acid model system described above was added to a mixture of 4.7 ml of 75% ethanol, 0.1 ml of 30% ammonium thiocyanate, and 0.1 ml of 2x10-2 M ferrous chloride solution in 3.5% HCL. After 3 min, the PV was measured by reading the absorbance at 500 nm following color development with FeCl2 and thiocyanate at different intervals during the incubation period of 7 days at 40Â±1Â°C.
Thiobarbituric acid (TBA)
The TBA method was used according to Kim et al. In this step a sample of 50 uL from the reaction mixture was added to a combination of 0.8 mL of distilled water. 0.2 mL of 8.1% sodium dodecyl sulfate, 1.5 mL of 20% acetic acid (pH 3.5) and 1.5 mL of 0.8% 2-thiobarbituric acid (TBA) solution in water. The mixture was heated at 100Â°C for 60 min. After the mixture was cooled and mixed using a vortex before read the absorbance at 532 nm and the antioxidative activity of the samples was expressed as malondialdehyde (MDA) concentrations.
Determination of ACE Inhibitory activity
The ACE inhibitor activity was measured by the method of Cushman and Cheung with slight modifications. Exactly 30l of 30 % (w/v) sample solution with 30 l of ACE solution (25 munits/mL) was pre-incubated at 37°C for 1 hour, and the mixture was incubated with 150l of substrate (8.3 mM Hip-His-Leu in 50 mM sodium borate buffer containing 0.5 M NaCl at pH 8.3) for 1 hour at 37°C. The reaction was terminated by the addition of 250uL of 1.0 M HCL. The resulting hippuric acid was extracted with 500ÂµL of ethyl acetate. After centrifugation at 2000 x g for 15 minutes, 700ÂµL of the upper layer was transferred into a test tube and evaporated. The hippuric acid was dissolved in 1.3mL of distilled water, and the absorbance was measured at 228 nm using a UV/VIS spectrophotometer (Kontron, UK). A positive control (without peptides), HHL control (without ACE and peptide) and captopril were also run in triplicate together with the samples (<2kDa, 5kDa, 10kDa, >10kDa). The ACE inhibitory activity of the samples and captopril were expressed as % ACE activity (equation 1):
The statistical analyses were performed with SPSS software (17.0).
Defatting sunflower seed
For defatting the kernels, two methods were used, hexane extraction and soxleht, Soxleht is a semi automated method and faster than hexane extraction, However Soxleht (it)operates at higher temperatures reaching up to 225°C which could easily denaturize helianthinin to its forms 11S 7S, and 2S. Evidences from differential scanning calorimetry(DSC) shows that 2S albuins structures were stables at 110°C at pH 7 and helianthinin at 105°C, 11S at 90°C and 7S at 65°C. As denaturation is when protein structure unflod to its primary structure however, there was a concern whether the high temperature would change the radical structures of amino acids affecting the ultimate result for antioxidation and ACE activity assessment.
Moreover, the results were compared with the label of the product as shown in table 2. Hexane sample was found have 49% of fat and soxleht 47, 5 % matching with the label hexane showed, but hexane showed a difference of 1.5% higher than the label.
As for the protein content Kjeldahl method was used, 6 raw samples plus four samples each 2 for hexane and soxleht. Hexane showed a total of 27.4% and soxleht 27.43% both after correction (see appendices for demonstrations of calculation), both results did not agree with the value on the label (19.8%). However, the result from the raw sample did match with both methods with a total protein content of 27, 8%.
These discrepancy may happen because the label is normally based on a one time estimative determined by a number of samples analyzed. However, there are differences from batch to batch, as they come from different regions of the world, for instance, different soil characteristics, environmental conditions such sun exposure, water availability and variety of seeds which may change the nutrient composition of the seeds.
Sunflower Isolate Preparation
The first of the series of extractions the mild acidic extraction was made in two steps as explained in the methods section.
The best protein isolate was from the alkaline extraction using sodium sulphite, followed by ethanol dephenolisation prior to alkaline extraction. which resulted in an acceptable clear color, therefore was the sample used for the rest of the study.
MECANISMO DE AÇÃO DO CAPTOPRIL
Como anti-hipertensivo, inibe competitivamente a ECA (Enzima Conversora da Angiotensina), diminuindo assim a conversão da angiotensina I em angiotensia II que é um potente vasoconstritor. A queda da angiotensina II leva a um aumento na atividade da renina plasmática (PRA) e a uma diminuição da secreção da aldosterona levando a um pequeno aumento de potássio e sódio e a uma maior eliminação de líquidos; inibidores da ECA reduzem a resistência arterial periférica e podem ser mais efetivos em hipertensão com renina alta.
Como vasodilatador na insuficiência cardíaca congestiva, diminui a resistência vascular periférica e a pressão intravascular pulmonar, aumentando o débito cardíaco e a tolerância aos exercícios. Absorção: gastrintestinal (75%); alimentos reduzem a absorção em 30 a 55%. Ação - início: 15 a 60 minutos; duração: 6 a 12 horas. Biotransformação: no fígado. Eliminação - urina: mais de 95% (não metabolizado: 40 a 50%; metabolizado: restante).