Determination of oleuropein of olive leaf using high performance liquid chromatography and optimizing the production of nano-structure lipid carriers containing it

As the demands for functional foods are on the rise, manufacturers have been seeking methods to use compound ingredients that make health promotion feasible. Nanoscience and nanotechnology have gained popularity in the 21st century. Scientists believe that nanotechnology can be used to handle matter at nano-dimensions. Nano powders, having a wide range of applications, were developed following the introduction of nanotechnology (Ananthu & Renjanadevi, 2016). It is believed that one the ways to save bioactive food ingredients from environmental damage and disguise their displeasure attributes is through encapsulation. Solid lipid nanoparticles (SLN) and nanostructure lipid carriers (NLC) are the novel types of nanostructures; researchers argue that they are the novel colloidal sensitive carriers that can be used for scientific and industrial applications (Pardeike, Hommoss, & Muller, 2009). Traditionally, the Mediterranean diet has been consisted of olive tree (Olea europaea) products that led to decreased risk of chronic diseases such as cardiovascular diseases and several cancers. Phenolic acids, phenolic alcohols, flavonoids, and secoiridoids are the usual phenolic compounds found in olives. Olives are rich in oleuropein, demethyloleuropein, and ligstroside (Yoon, 2018). Several pharmacological features have been reported in oleuropein such as antioxidant (Visioli, Poli, & Galli, 2002), anti-inflammatory (Visioli, Bellosta, & Galli, 1998), anti-atherogenic (Carluccio et al., 2003), anti-cancer (Owen et al., 2000), antimicrobial (Tripoli et al., 2005), and antiviral ones (Fredrickson & Group, 2000); hence, it has been used commercially as food supplement in Mediterranean countries. Besides, research shows that oleuropein exerts cardio-protective effect against acute adriamycin cardiotoxicity (Andreadou et al., 2007); it was also reported that they are involved in anti-ischemic and hypolipidemic activities (Andreadou et al., 2006). Oleuropin is the most abundant type of phenolic compounds in olive leaf and its therapeutic effects are well known as a potent antioxidant. Extraction of oleuropein from olive leaves as a rich source of this compound is very valuable. On the other hand, the encapsulation efficiency of this compound is an effective way to maintain its characteristics during storage. The present research tried to investigate the synthesis method, characterization, and physical stability of nanostructured lipid carrier containing of olive leave extract. Various methods including high mechanical shear, high-pressure melt-homogenization method can be applied to synthesize the novel nanoencapsulation system (NLC). Since data on the effects of formulation compound on physicochemical properties of oleuropein-loaded NLC is scarce, the present research attempted to study the effect of concentrations of solid and liquid lipid, surfactant and olive leaf extract powder on physical and chemical characteristics of oleuropein-NLC by the application of one-at-time method. Initial component concentrations were chosen by the Design Expert Software (version 7.0.0) and one-factor-at-a-time method/one-variable-at-a-time method at various levels. In a study an extract containing oleuropein, was extracted from olive leaves, was prepared in the form of lipid nanocarriers. Extraction was performed by combining solvents ethanol: water (70:30) in a warm bath (40 °C for 30 minutes) using an HPLC apparatus. By designing 15 formulas for the nano-structured lipid carrier, the particle size and encapsulation efficiency were determined by a zeta sizer and HPLC, respectively. After determining the optimal formulation, zeta potential was also evaluated using a zeta sizer. The results showed that the extraction method of oleuropein from olive leaf with ethanol and water (70:30) contained 221.37 mg/g of oleuropein with a purity of oleuropein 137.22. The large amount of olive leaf extract in extraction with ethanol could be due to the better solubility of the active compounds (polyphenols) in organic solvents, such as ethanol. Result is showed the average particle size for all freshly produced oleuropein-loaded NLC formulations with diameters of ~0.10±0.00 and 5767± 640.15 nm. Also, the results of the dynamic light scattering test showed that the optimal formulation had a mean particle size of 12.19 nm, a scattering index of 0.153, a zeta potential of -43.3-millivolt. The negative loads on the surface of nano-carriers were probably due to the presence of lipophilic surfactants with negative load (lecithin) and free fatty acids (linoleic acid and linolenic acid, oleic acid, and free fatty acids in soybean oil). Probably the hydroxyl group in the encapsulant (oleuropein) has a hydrogen bond with a choline group in the polar head of the phosphatidylcholine and the choline group with the positive load is pulled inside and the phosphatidyl group with negative load is driven to the membrane surface. Therefore, the negative load of the nano-carriers is increased, which leads to electrostatic disposal of particles and, finally, stability of these particles. Microencapsulation values of nano-carriers varied from 1.27 ± 0.10 to 82 ± 0.41 and the formula with code 12 had the highest efficiency of microencapsulation. Adding liquid lipid to the lipid mixture and increasing the liquid part to solid part ratio resulted in the formation of a more amorphous state and less crystalline state of the nano-carriers and, finally, led to the defective and increased encapsulation material in nano-carriers (increased efficiency of microencapsulation). This could be probably due to more solubility of the encapsulation material in the liquid part of the lipid. Finally, the results of the encapsulation efficiency showed that the nano-carrier had 82% of the overlapping oleuropein. One of the aims of the application of nanocarriers in the food industry is to improve bioavailability, cover the undesirable odor, flavor, color, and improve solubility of some materials. Results of this work depicted that adding nanocarriers containing olive leaf extract powder as the source rich of oleuropein to the food, such as sauce, could improve the undesirable appearance, color, odor and flavor of the extract powder (polyphenols such as oleuropein) and also enjoy high durability of antioxidant properties of the polyphenols existing in the nanocarrier. A practical opinion/idea is that encapsulation of olive leaf extract powder as nutraceutical component can be applied to nutritive and pharmaceutical industries as well as production high quality products with whiteness, uniformity/homogeneity, appropriate flowability and flavor properties. Finally, the nano carrier with the lowest olive leaf extract powder, had the highest encapsulation efficiency, the lowest dispersity index and particle size.