# Nanoformulation of Peptides for Pharmaceutical Applications: In Vitro and In Vivo Perspectives
## 2 Overview of Peptides
### Recent Methods for Peptide Extraction and Formation  
The extraction of peptides from several unique sources has increased in recent times. Peptides can be extracted from the proteins of milk and other sources via fermentation and other methods, as shown in Figure 1. In addition, there is a need for techniques to maximize peptide activities in food systems as well as optimize their use in pharmaceutical applications [46]. The isolation and purification of peptides involve the formation of continuous and automated systems. Thus, extensive research is required for designing specific column chromatography methods that can substitute earlier methods, such as salting out or solvent extraction. The advanced column chromatographic methods will provide peptides with optimized parameters and low degradations to enable their integration in certain nutraceuticalal and pharmaceutical applications [47]. Enzymatic hydrolysis, microbial fermentation, and chemical synthesis are the major methods for the production of peptides. Enzymatic hydrolysis is considered the most viable and dependable method for peptide production with intact functions, such as antioxidant properties. Hence, this method is utilized for the formation of a wide spectrum of peptides as well as peptide hydrolyses from potatoes, whey, and corn. This method has been used over the decades and it is still commonly used [49; 50]. Some examples of enzymes used in this method are alcalase, neutrale, pepsin, and others [51; 52; 53]. The antioxidant abilities of peptides and peptide mixtures depend on the method of hydrolysis used, among other factors (the type of proteases used, protein source, and pretreatment of protein substrate). Both crude and pure enzymes can be applied for the extraction of peptides with antioxidant activities. Nevertheless, crude protein mixtures are used to minimize the cost of production [13; 54]. Further, peptides can also be extracted via fermented milk protein products using protease. Enzymes from the bacterial strain named _Streptococcus thermophiles_ were identified to be beneficial for the extraction of peptides from bovine caseins [55]. The actions of proteases from _Bacillus amyloidateiens_ and _Bacillus subtilis_ A26 on Thornback ray skin gelatin were also utilized for the extraction of peptides with antioxidant and acetylcholinesterase inhibitors activities, respectively [56]. In another study, proteases from fungal-digested extracts of meat protein were demonstrated to have potential for extracting peptide mixtures with remarkable acetylcholinesterasecholinesterasease and antioxidant activities in an in vitro model [57]. Microbial fermentation is another method for the production of peptides. The development of peptides with antioxidant activities via the fermentation process by microbes is commonly used by the food industry. Fermented soybean substances, such as tempeh and natto, were reported to contain peptides with antioxidant properties [58]. Peptides with antioxidant activities can also be produced from douchi (soybean product) via fermentation using fungal cultures [13; 58]. Foods, such as yogurt with vital peptides, can be developed from milk protein through the actions of proteolytic systems [59]. The fermentation of navy bean milk with _Lactobacillus helevticus_ MB2-1, _Lactobacillus plantarum_ 70810, _Lactobacillus bulgarcius_, and _Lactobacillus plantarum_ B1-6 showed greater acetylcholinesteraseinesterase activity, compared to untermented mil [60]. Similarly, the fermentation of milk with _Bifidobacterium bifutum_ MF 20/5 has led to the production of milk products with higher acetylcholinesterasecholinesterasease inhibitory activity. Other peptides, such as LVYPF (inhibbits acetylcholinesterasease), LPLP (inhibits acetylcholinesterasease), and VLPVPQK (scavenges free radical) have also been isolated from fermented milk [61]. Chemical synthesis is an essential method used for developing peptides. This method is relatively simple, inexpensive, and it involves the use of an alkaline or acidic solution.