Analytical studies on bioactive components of natural materials

Abstract

Since historical eras, plants have been considered the most valuable resource in nature due to their high nutritional value as food and their healing properties. Many plants are classified as herbal medicines as a result of their use as traditional remedies. The various healing properties of plants have been discovered and documented over time, and these medicinal plants have been used for the treatment of heart diseases and skin problems, lowering high cholesterol and sugar levels in the blood, and also curing cold-like symptoms. Today, the demand for medicinal plants persists. These plants not only grow in the geography where they originated, but they have propagated across many parts of the world. Plants are rich in phenolic compounds, and carotenoids, as well as vitamins and minerals, and they mostly have antioxidant and anticancer effects. A diet rich in plants is crucial for maintaining health, but identification and isolation of the bioactive components are also necessary for the production of new products and drug formulations, as encouraged by the World Health Organization (WHO). Türkiye has different climates and ecological conditions in different regions, which leads to a rich plant flora. Thus, the determination of the bioactive constituents in local medicinal and aromatic plants is especially significant and contributes to their commercial and export value. Determination and separation of the active ingredients and components in medicinal plants, herbs, and spices have been studied by using many analytical techniques. A relatively new but also very powerful technique, capillary electrophoresis (CE), which was introduced in the 1980s, facilitates studies in this field. Diminution of the sample and solvent consumption, very short analysis time, and high resolution are highlighted as the advantages of CE. The separation principle depends on the formation of a bulk flow in capillaries under high voltage during separation, where all analytes separate within the column according to their relative motion. Moreover, traditional medicinal plants and some plants with therapeutic effects attract great attention as a good source of additives in food products and the development of some food packaging materials. Large polymeric molecules derived from living organisms or from their products are called biopolymers, which are extensively used for this purpose due to their non-toxic nature and biodegradability. Nowadays, the development of novel materials with desirable properties is achieved by using biopolymer blends, and enhancement of the protective properties of the materials with the addition of natural extracts and products has become a highly investigated research area. The studies are focused on limiting the use of plastics and diminishing waste accumulation; however, replacing them with safer alternatives and increasing food protection with natural additives also meet consumers' demands. In the first part of the thesis study, two important bioactive components khellin and visnagin amounts were determined in 5 different plant parts of Ammi visnaga L. and Ammi majus L., two plants used as traditional herbal medicine. A micellar electrokinetic chromatography (MEKC) method was successfully applied and completed in 8.5 minutes. Separation conditions were optimized, 20 mM borate, 20 mM SDS and 5% (v/v), and pH 9.6. Detection of the species was carried out with a UV detector at 245 nm wavelength. There are a limited number of studies on the determination of khellin and visnagin from A. visnaga and A. majus flowers. However, this study is the first report on the determination and comparison of the khellin and visnagin contents of these two plants grown in Türkiye in their five different parts, namely: flowers, leaves, roots, seeds and stems, as well as the determination of the antioxidant properties applied to the plant extracts. Linear calibration curves were constructed and regression coefficients were calculated as 0.997 and 0.998 for khellin and visnagin, respectively. The applied method was successfully validated, and the amounts of khellin and visnagin in the samples were calculated in the range of 1.60 to 22.60 mg g-1 dry weight of the plant. The limit of detection (LOD) of the method was calculated as 0.83 mg L-1 for khellin and 0.99 mg L-1 for visnagin. Visnagin was found only in A. visnaga and its amount was found to be between 5.80 and 18.50 mg g-1 dry weight of the plant. The antioxidant power of the extracts was determined by the DPPH method, which measured the ability to react with the 2,2-diphenyl-1-picrylhydrazyl radical, and the highest effect was found in the flowers of both species, followed by the seeds and leaves. In the second part of the thesis, a capillary electrophoresis method was developed to determine the amounts of rosmarinic and carnosic acids, in 14 Salvia species obtained from various regions of Anatolia. There are numerous literature studies on the determination of Salvia ingredients grown in various regions and countries. However, this study covers 14 Salvia species growing in Anatolia were examined and the correlation between the amount of bioactive species and antioxidant properties was reported. Optimized separation conditions were 20 mM borate and pH 9.6. A silica capillary column with an effective length of 59 cm was used for separations, and the detection of the analytes was carried out with UV detection at a wavelength of 210 nm. The migration time for carnosic acid was 4.1 minutes, while for rosmarinic acid was 5.8 minutes. Linear calibration curves were constructed for both analytes and the regression coefficients were calculated as 0.998 for rosmarinic and carnosic acids. The detection limit of the method was calculated as 1.86 mg mL-1 for rosmarinic acid and 1.72 mg mL-1 for carnosic acid. Recovery studies were carried out for the validation of the method, and the values were found to be between 94.8% and 101% for rosmarinic acid and 90.2% and 96.7% for carnosic acid. While rosmarinic acid contents in Salvia species were found to be between 1.08 and 18.70 mg g-1 dry plant, carnosic acid was found to be 11.00 mg g-1 dry plant in only one Salvia specie. The antioxidant power of the extracts was determined using the iron (III) ion-reducing antioxidant power method (FRAP) and the DPPH method, in which the 2,2-diphenyl-1-picrylhydrazyl radical is used and the ability of the studied substance to undergo radical reactions is measured. The Follin-Ciocalteu method was used for total phenolic substance determination. The correlation between rosmarinic acid content and DPPH method results is 0.77, and the correlation between rosmarinic acid content and FRAP method results was found to be 0.82. In the third part of the thesis, vinegars which were obtained by fermentation of various fruits were used as safe and non-toxic solvents to produce chitosan-based, non-toxic, biodegradable materials with improved properties. Since chitosan is a natural biopolymer and obtained by the deacetylation of chitin, which is abundant in the shells of crustaceans, and dissolves in dilute solutions of organic acids, films were prepared by dissolving 1% (w/v) chitosan in vinegar and water ratio of 1:1 (v/v). Chitosan was also preferred due to its antimicrobial and non-toxic properties, and its physical and mechanical properties are compatible with the desired coating materials. In this study, kinds of vinegar produced from pomegranate, apple, grape and hawthorn fruits were used. The spectroscopic, physical and mechanical properties of these films were compared with each other. It was observed that vinegar films had the ability of blocking ultraviolet rays in the range of 200 to 350 nm, and especially the increase in the elasticity of the film prepared with pomegranate vinegar was obtained. An 11-fold increase in the antioxidant power of the film prepared with pomegranate vinegar, determined by the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) method, was observed compared to the control film i.e. chitosan film prepared in commercial acetic acid. It was also determined that prepared films showed a higher antimicrobial effect against gram-positive and gram-negative bacteria, compared to the control film. Materials prepared with four vinegar varieties showed improved antimicrobial, antioxidant, optical and elastic properties, and they are promising in the application of these materials as potential and economical food packaging materials. In the fourth and last part of this thesis, a potential biopolymer-based food packaging material was produced by using the extract of the purple basil (Ocimumbasilicumpurpurascen). Aromatic basil leaves, known for their antioxidant, antiviral and antibacterial properties, are rich in phenolic compounds, minerals, vitamins, and anthocyanins, which are the pigments responsible for the strong purple color of the plant. The chemical structures of the anthocyanins change with pH variation, and as a result, color changes are observed. Thus, the prepared material is non-toxic and biodegradable, and it can be used as a natural indicator for the determination of food freshness. In order to provide the desired physical properties of the material, pectin and alginate biopolymers were used together. Films were prepared by mixing 1% (w/v) pectin and 0.5% (w/v) alginate with purple basil extract in 1:1 (v/v) water in a total volume of 30 mL for 5 hours. The polymer mass was formed. It was prepared by adding 1:1 glycerol by mass and dissolving it, then pouring it into petri dishes and drying it. In order to prevent the obtained biopolymeric films from dissolving in water, the films were cross-linked with CaCl2 solution. The optical, spectroscopic and physical properties of the obtained material (swelling, water solubility, water vapor permeability, moisture content analysis), antioxidant and antimicrobial properties, and the release of anthocyanins over time were evaluated. In addition, it was tested with a real food sample to see if this material could detect food spoilage as expected. The resulting material showed high antioxidant properties and antimicrobial power against both gram-positive and gram-negative bacteria and was observed to block ultraviolet light. It has been proven that its physical properties are close to the values stated in the literature for these materials and that it is a potential food packaging material with a potential natural indicator feature, with the color change that accompanies food degradation, which is also accompanied by pH change.