Project summary
Zein is the main form of protein storage from corn which is the largest and most important agricultural commodity worldwide and has been investigated for different uses (food industry, pharmaceutical industry, as drug delivery systems, etc.). Biologically, zein is a mixture of different peptides of various molecular size and solubility. Particularly, zein due to the large amount of nonpolar amino acids, can form aggregates and interact with drugs by forming stable protein–drug complexes and therefore, it can delay the release of the specific drugs. The main goal of the proposed project is the extraction of α-zein from autochthonous corn seeds (using eco-friendly solvents and procedures), purification, characterization and the usage of α-zein to synthesize new zein based conjugates. New and very accurate analytical methods will be used, such as: HPLC, MALDI-ToF mass spectrometry, SDS-PAGE, FT-IR or UV-vis spectroscopy.
Objectives
I.1. To improve both extraction and purification of α-zein from autochthonous corn seeds, using different methods. Because a high amount and quality of sample homogeneity is required to perform structural studies and to be used in further synthesis. Achieving the proposed objectives will require the use of some methods such as: high-performance liquid chromatography (HPLC), mass spectrometry (MS), electrophoresis (SDS-PAGE), infrared spectroscopy (FTIR) and UV-vis spectroscopy;
I.2. To synthesize and characterized new zein conjugates (functionalized with histidine, zeins conjugated with salicylic acid or other drugs);
I.3. To validate in laboratory environment the conjugated zein used for retention of heavy metals from human body and for controlled drug release which may lead to new biomedical applications of zein;
I.4. To obtain new experimental data which can be published in high impact factor journals and presented at domestic and international meetings.
Results
I. Optimization of zein extraction and zein characterization
Zein, a complex protein, is one of the few grain proteins that can usually be isolated in a considerable amount from corn maize. The process of extracting zein proteins from the hybrid strain began with grinding corn seeds, from which two fractions with 710 µm and 250 µm granulations size were selected for further studies. Both maize four, 710 µm and 250 µm granulations were suspended in two separate aqueous ethanol solutions, 65% and 95%, respectively, and the resulted samples were notated 65/250, 65/710, 95/250, and 95/710. Table 1 shows the efficacy of 15 min of extraction using either the ultrasonic bath or the ultrasonic homogenizer.
Our findings suggest that a combination of SDS-PAGE and high-resolution mass spectrometry is crucial for characterizing polymorphic protein composition in one dimensional gel band patterns by providing a better characterization of individual protein isoforms.
For a precise identification of extracted zein, SDS-PAGE electrophoresis was followed-up by in gel digestion, MALDI-ToF, and electrospray ionization (ESI) mass spectrometry analysis. Using ESI mass spectrometry, we analyzed the zeins extracted with 65% and 95% ethanol from the maize inbred KWS 3381 flour with the particles size 250 μm and 710 μm. As an example, the mass spectrum shown in Figure 2 corresponded to the zein extracted using an ultrasonic homogenizer with 95% ethanol from the 710 μm maize flour and displays peaks at m/z 974.31, 1016. 59, 1062.6, 1113.12, 1168.89, and 1230.12 that were assigned as multiple charged ions of 19 kDa zein PMS2 (Uniprot accession no. P24450).
II. Synthesis of zeine-based conjugates
To study the ability to bind different metal ions or to release drugs, zein functionalized with histidine and zein conjugated with salicylic acid were synthesized.
III. Investigation of zein-based conjugates used as controlled drug release systems
The results obtained following the synthesis of zein-based conjugates with salicylic acid show the importance of this corn protein and also justify further investigations. Moreover, from a biomedical perspective, zein conjugates could be used as drug delivery devices if a thorough investigation of the interactions between this protein and the active compound is established.
