Investigation and isolation of peptide based antiglycating agents from various sources

Introduction

Non-enzymatic glycation occurs through the reaction of reducing sugars with free amino groups of proteins. Initially the product called Schiff’s base is constituted. Later more stable products called Amadori compounds are formed which constitution is followed by advanced glycation [1]. Recent studies have shown that formation of advanced glycation end products (AGE) is autooxidative process dependent, affecting the reactive oxygen species (ROS) formation [2], [3], [4], [5], [6]. Accumulation of AGE products alters the structure and function of tissue proteins and as a result this takes part in aging, aging dependent diseases, and pathogenesis of important diseases [7], [8], [9], [10]. For this reason, prevention of non-enzymatic glycation and formation of AGE products have become a prior interest in recent years. Various endogenous and exogenous resource based compounds have been mechanistically evaluated in this regard. Especially dipeptides such as carnosine and serine analogs, compounds such as pyridoxamine, aminoguanidine, pyridoxal-5-phosphate, and n-acetyl cysteine have been evaluated for their antioxidant, metal chelating, and deglycation activities [10], [11], [12], [13], [14], [15], [16]. For this regard, obtainment of most possible potent compounds with metal chelating and deglycating activities. For this aim, isolation of especially peptide based compounds from histidine and cysteine rich protein sources was aimed.

Materials and methods

Dried plants seeds were commercially ordered and then identified in Ege University Department of Biology – Botany. All the chemicals used in this study are of analytical grade.

Extraction of peptides from various sources

All plant species used in this study [Soybean (Glycine max), Barley (Hordeum vulgare), Wheat (Triticum aestivum), Oat (Avena sativa), Almond (Prunus dulcis) and Walnut (Juglans regia)] were minced until the material became powder. For each sample, 1 g of sample were mixed with various solvents (20 mM sodium acetate pH 9, 20 mM sodium acetate pH 5, 5% TCA, 1 M acetic acid in 10% acetonitrile.) and stirred at room temperature for 1 h at 175 rpm. Mixtures were homogenated and filtered through glass wool under vacuum. The extracts were centrifuged at +4°C for 15 min at 9000 g. Cold acetone was added to the mixture at a 1:1 (v/v) ratio and centrifugation was repeated.

Inhibition of in vitro protein glycation

The methodology was based on that of Wu and Yen [17]. BSA (10 mg/mL) was incubated with glucose (500 mM) in phosphate buffered-saline (PBS) (3mL total volume, pH 7.4) and extract containing 0.02% sodium azide at 60°C. All the reagent and samples were sterilized by filtration through 0.2μm membrane filters. The protein, the sugar and the prospective inhibitor were included in the mixture simultaneously. Aminoguanidine was used as an inhibitor positive control. Reactions without any inhibitor were also setup. Each solution was kept in the dark in a capped tube. After 2 h of incubation, fluorescence intensity (excitation wavelength of 370nm and emission wave-length of 440nm) was measured for the test solutions.

% inhibition values were calculated according to the following formula.

% inhibition=[(Acontrol−Ablank)/Acontrol]×100

Determination of IC50 values of peptide extracts

The solvent system that yielded the highest deglycation was chosen for extraction of all plant samples. For the determination IC50 values of all peptide isolates, inhibition assays were done at five different protein concentrations with three replicates. Results were compared in percentages of glycation and inhibition. Using the inhibition results and linear regression formulations, IC50 value was calculated as the protein concentration that inhibits the 50% of the glycation activity. IC50 values for carnosine and aminoguanidine were determined too for comparison.

2,4,6-Trinitrobenzenesulfonic acid (TNBS) method

For determination of free amino groups in proteins and other molecules, various methods such as TNBS and o-Phthalaldehyde (OPA) are used. TNBS method was chosen for its sensitivity, for detection of peptides [18]. The peptide isolates were dissolved in hot 1% sodium dodecyl sulfate. A sample solution (0.250 mL) was mixed with 2 mL of 0.2125 M sodium phosphate buffer (pH 8.2) and 2 mL of 0.1% TNBS and the mixture was incubated in dark for 60 min at 50°C. The reaction was quenched by adding 4 mL of 0.1 N HCl and the absorbance was measured at 340 nm using Multiskan™ FC Microplate Photometer.

Total thiol groups assay

Thiol groups of the peptide preparates were analyzed spectrophotometrically via Ellman method [19]. Reagent mixture contains 10 μL sample, 180 μL 0.1 M pH 8.2 Tris-HCl, and 100 μL Ellman reagent prepared in the same buffer (60 mg 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB)/100 mL). The absorbance of yellow colored product of the reaction, 2-nitro-5-mercapto-benzoic acid, was measured at 405 nm via microplate reader (Thermo Scientific™ Multiskan™, FC, USA). Glutathione (GSH) standard with a concentration range of 2–20 μg/mL was used for calculation total thiol of the samples.

Determination of metal chelating activity

To determine the metal chelating capacity of the peptide isolates with deglycation activity, copper-chelation assay [20]. Copper (II) sulfate was dissolved in 10 mM KCl containing 30 mM hydroxylamine hydrochloride buffer pH 5. One milliliter copper solution (3 mM) and 400 μL 1 mM murexide is added on 1 mL of different dilutions of samples. The mixture is incubated 3 min at room temperature. Proportion of absorbance at 485 nm to absorbance at 530 nm gives the metal chelation activity. Using a copper (II) sulfate standard with a range of 0.025–0.125 mM, free copper amounts were calculated. Assays were done in three replicates.

%Chelation (bound copper)=[(Fcontrol−Fsample)/Fcontrol]×100

Determination of total antioxidant capacity

This assay is based on reduction of copper (II) to copper (I) by antioxidants [21]. Ten millimeter CuCl₂·2H₂O, 7.5 mM neocuproin, and 1 M ammonium acetate pH 7 were respectively added to the wells of microplate, each with an equal volume of 50 μL. Mixtures were preincubated for 15 min at 37°C and absorbance was measured at 450 nm. Ascorbic acid was used as a standard, with a concentration range of 20–200 μmol/mL. Assays were done in three replicates.

Investigation of peptide isolates by RP-HPLC

The peptides were eluted by a linear gradient from 98% to 2% solvent A (0.1% trifluoroacetic acid (TFA) in deionised water) in solvent B (0.1% TFA in 90%, v/v, acetonitrile in deionised water) over 20 min. Separations were conducted at 40°C at a flow rate of 1 mL/min. The eluted peptides were detected at 215 nm.

Results and discussion

Diabetes is a multifactorial disease which affects world population at an increasing rate. For this reason, aside from preventive life styles, treatment based approaches are also arising. It is known that extended periods of hyperglycemia increases non-enzymatic glycooxidation based damage to protein structure and function, resulting in protein aggregation based diabetic complications and take role in diseases such as Alzheimer’s disease, cataract formation, rheumatoid arthritis, neurodegenerative diseases, and pathogenesis of important diseases [22], [23], [24]. For this reason, approaches for minimization of non-enzymatic glycation and formation of advanced glycation end products are of prior importance. Studies regarding compounds with deglycation activities especially for carnosine and its analogs are present in the literature [25], [26], [27], [28], [29]. But currently compound characterization studies for deglycation are mainly limited for natural sources. These studies have mainly involved characterization of total plant extract for deglycation activity [30], [31], [32], [33]. In this study, plant species with proteins of high cysteine and histidine contents (Glycine max, Hordeum vulgare, Triticum aestivum, Avena sativa, Prunus dulcis and Juglans regia) were used for extraction of endogenous peptides. Determination of the sample with the highest biological activity and its further characterization was aimed. This study is expected to be preliminary for the future studies regarding the discovery and characterization of peptide based deglycating compounds and to reveal possibly effective AGE peptide inhibitors as the main outcome.

Peptide extraction from various sources

First the extraction method was optimized to obtain bioactive compounds with highest yield. Four different extraction conditions were tried. The prepared extracts were vacuum dried yield was calculated to be the dry weight obtained per gram of plant sample. Using four different extraction conditions, increasing protein concentration linearly decrease AGE formation for extracts prepared with sodium acetate pH 5 buffer and TCA. Extracts prepared with sodium acetate pH 9 buffer and acid/acetonitrile solvent did not increasingly inhibit the glycation at higher protein concentrations than 40 μg/mL.

According to the results, the TCA extraction method that yielded the lowest IC50 value for deglycation was selected for further studies.

Determination of deglycation activities and other properties of peptide isolates

All the isolates were dissolved in appropriate solvent according to the downstream assays. First, effect of protein concentration on AGE formation were studied (Figure 1).

Figure 1:

Curves and linear regressions of AGE inhibition activities of (A) walnut peptide extract, (B) almond peptide extract, (C) wheat peptide extract, (D) oat peptide extract and (E) barley peptide extract.

Inhibitory activity results have shown that aside from the walnut derived one, every peptide isolates was potent for deglycation. Moreover, oat, barley, wheat, and soya derived peptide isolates were more potent inhibitors of AGE formation than the standards aminoguanidine and carnosine. IC₅₀ values of the peptide isolates were given in Table 1.

It was observed that soy peptide isolate has the highest antioxidant activity, metal chelating capacity, and free amino and thiol group content when compared to the other plants those were used in this study (Table 2). Oat and barley hydrolyzates are also have similarly strong activities. When compared to the known deglycating and antioxidant compounds carnosine and amino guanidine, almond and wheat peptide isolates were stronger, with higher free amino and thiol group content and metal chelating capacity. On the other hand, almond and barley peptide isolates were surpassed by soy and oat peptide isolates.

The alleged mechanism of prevention of glycation by these compounds are thought to be caused by interference of the compounds to Schiff’s base formation, thus blocking AGE formation. As a result, five peptide isolates (soy, barley, almond, wheat, and oat) are thought to have remarkable deglycation activities. Among them, especially soy peptide isolate is considered to be valuable in terms of antioxidant activity and metal chelating capacity too. RP-HPLC separations of the soybean peptide isolate was performed in order to analyse the complexity of the peptide preparate, purification and enrichment of more potent peptides among the isolated pool and initiate structural studies. For this purpose, a sample chromatogram post optimization of separation studies is shown in Figure 2.

Figure 2:

HPLC profile of the soybean isolate (Supelco ODS C18 (4.5×250), gradient: 98% A (0.1% TFA), % 2 B (Acetonitrile/0.1% TFA), 20 min 60% B, 1 mL/min).

Examination of the chromatograms taken at three different wavelengths by DAD detector were examined and it has been observed that the sensitivity was higher in the chromatogram in which the peptide bonds were measured. The eluate collected between the 4th–5th min of the run showed a dense peak. The collected fraction has exhibited 74% AGE inhibitory activity.

Conclusion

Bioactive proteins and peptides are recently among important research topics. Studies have revealed that bioactive peptides take part in many important physiological processes, which has lead to discovery of new peptides and chemical synthesis of them. Development of new bioanalytical techniques have further facilitated discovery of a hormones and neuropeptides. Solid phase and classical solution phase techniques allowed chemical synthesis of oligopeptides. This allowed rapid and high fidelity production of bioactive peptides when compared to the molecular biological and genetic engineering techniques, which involve in vivo synthesis and demand thorough purification. Through these developments, important steps were taken for explanation of biochemical events and drug development studies. In this study, isolation and obtainment of bioactive peptides were of priority. Briefly, results have shown that respectively antiglycation activity of soya, barley, wheat, and almond significant when compared to their standards carnosine and aminoguanidine. Soya peptide isolate has shown the highest deglycation, antioxidant, and metal chelating activities, providing important results for development of more potent and specific inhibitors. Purification and structural analysis of products those inhibit AGE formation along with use of bioinformatics tools, similar peptides can be discovered or designed, ultimately resulting in a wider repertoire of products in this field. In vivo pharmacokinetic studies will also be required as further characterization steps.

It is clear that this study provides a valuable preliminary data to literature and can become a template for choosing a starting material for the design of compounds with desired bioactivities such as metal chelation, antioxidant, deglycation, etc. for advanced researches.