Bohdanovych T. Development of biotechnology for obtaining compounds with anti-inflammatory and antioxidant properties from the hairy roots of Artemisia tilesii Ledeb.

Thesis to fulfill requirements for Doctor of Philosophy degree in the specialty 091 Biology (09 Biology). – Institute of Cell Biology and Genetic Engineering NAS of Ukraine, Kyiv, 2024. The dissertation is devoted to the initiation of hairy roots with a high content of flavonoids, antioxidant, reducing and anti-inflammatory activity; stimulating the synthesis of polyphenolic compounds and obtaining of dry extracts from wormwood roots, which have the biological activities listed above. Via genetic transformation using Agrobacterium (Rhizobium) rhizogenes it is possible to obtain hairy roots that can grow indefinitely on nutrient media without the addition of growth regulators and synthesize bioactive compounds in quantities far exceeding such in parent plants. Wormwood plants belong to medicinal plants. However, the species Artemisia tilesii Ledeb. is still poorly studied. These plants have a limited growth area, are adapted to extreme conditions of existence and can be a source of biologically active compounds. That is why, the work was aimed at obtaining flavonoid-containing extracts with anti-inflammatory and antioxidant properties from the hairy roots of Tilesius’ wormwood, previously received by the author. Such extracts can be used to create new drugs with antioxidant and anti-inflammatory properties. The aim of the work was to create hairy roots of A. tilesii and obtain compounds with anti-inflammatory and antioxidant properties from their extracts. To achieve the goal, following tasks were outlined and solved: 1) carry out genetic transformation of Tilesius’ wormwood with a wild agropine strain of Agrobacterium rhizogenes A4; 2) screen hairy roots using biochemical methods; 3) analyze the relationship between the activity of the rolB, rolC, CHS (chalcone synthase) and PAL (phenylalanine-ammonia-lyase) genes with the growth rate of different lines of hairy roots and their accumulation of flavonoids; 4) determine the influence of cultivation conditions on the growth of hairy roots, synthesis of flavonoids and antioxidant activity; 5) to optimize the method of obtaining extracts with anti-inflammatory and antioxidant properties. The following research methods were used in the work: biotechnological, biochemical, molecular genetic, statistical data analysis methods. The scientific novelty of the obtained results is as follows. For the first time: 1) comprehensive analysis of the obtained lines of A. tilesii hairy roots was carried out, in particular, differences in the content of flavonoids, antioxidant activitiy and reducing power, hydrogen peroxide content, superoxide dismutase and catalase activities were determined, and a comparison of the features of the lines initiated using the wild A. rhizogenes strain and those with heterologous ifn-α2b and nptII genes was carried out; 2) content of phenolic acids in samples of the hairy roots of the Tilesius’ wormwood was compared using the HPLC method; 3) direct correlation of the activity of the rolB and rolC genes with the growth rate of the hairy roots of A. tilesii and an inverse correlation of the activity of PAL with the content of flavonoids was proved; 4) the influence of phenylalanine, light and methyl jasmonate on the growth of A. tilesii hairy root cultures, flavonoid synthesis and antioxidant activity was determined for the first time; 5) the presence of antiviral and anti-inflammatory activities in extracts of the hairy roots of A. tilesii was shown for the first time; 6) method of obtaining flavonoid-containing extracts from hairy wormwood roots using two-stage cultivation and elicitation with methyl jasmonate was optimized. Practical significance of the obtained results: 1) the obtained lines of hairy roots, which showed antioxidant, reducing, antiviral and anti-inflammatory activity, can be a source of valuable biologically active compounds; 2) the dry extract obtained from such roots can be used as a basis for the production of bio-safe preparations from plant raw materials; 3) the optimized method of obtaining a bioactive flavonoid-containing complex allows the effective use of roots and the scaling of the process by cultivating roots on a hormone-free liquid medium in bioreactors. The results of theoretical and practical research may be of interest to the pharmaceutical and medical industries, as well as to biotechnologists and industry representatives who are interested in intensifying the production of compounds with a wide range of biological activities. Keywords: Artemisia tilesii, Agrobacterium (Rhizobium) rhizogenes, rol genes, hairy roots, flavonoids, secondary metabolism, antioxidant activity, anti-inflammatory activity, reducing power, antiviral activity, nanoparticles, methyl jasmonate, elicitors, bioactive extract.