Analysis, genomic characteristics and biological activities of secondary metabolites of some Salvia species

Salvia (Sage) species belong to the families of the Lamiaceae (Labiatae) family, rich in terpenic plants. There are more than 900 Salvia species in the world, the main countries of which are China, Brazil, Mexico and mostly European countries in the Mediterranean region. Secondary metabolites of Salvia species in Turkey, represented by more than 105 taxa and 96 species, are mainly terpenoids, steroids, flavonoids and other phenolic compounds. Due to the presence of di- and triterpenes, they have anti-inflammatory, antiviral, hepatotoxic and cytotoxic/antitumor activities along with antioxidant, antimicrobial, cytotoxic, etc. properties of flavonoids. Salvia divinorum plant, in contrary to European and Asian species, is rich in diterpenoid compounds having neo-clerodane structure, especially salvinorin A and salvinorin B. It shows selectivity toward Kappa-opioid receptors (KOR) and is distinguished from other species due to its hallucinogenic properties. Screening the hallucinogenic active ingredients in plant species consumed by people in our Country is extreamly important since neo-clarodane diterpenoid compounds are common in Salvia species. Over 50 years, many neo-clerodane compounds have been isolated from species such as Lamiaceae, Verbenaceae, Euphorbiaceae, and Compositae (Asteraceae). Neo-clerodane diterpenes are known to be taxonomic markers in diterpenes due to the additional furan ring in their structure. Salvinorin A is a very strong hallucinogenic compound and, in combination with salvinorin B, displays selectivity toward Kappa-Opioid receptors of brain. The kappa-opioid receptor ligands play a role in mental disorders such as addiction, depression, mental disorder and schizophrenia. In this thesis, which is the continuation of our studies on Salvia species, 40-45 Salvia species collected from different regions of our country, and they were subjected to; i. Determination of the phenolic and salvinorin content of major secondary metabolites by a reliable method, using liquid chromatography / tandem mass spectrometry (LC-MS / MS). ii. Investigation of volatile organic compounds from other secondary metabolites by thermal desorption gas chromatography mass spectrometry (TD-GC-MS) iii. Analyses of genomic DNA sequences of Salvia species to generate genomic maps, identification of phylogenetic similarities between species, iv. Investigation of antioxidant and antimicrobial activities using different methods and establishment of their structure-activity relationship with the secondary metabolites they contain. In this PhD study, methanol extracts of arial parts of some Salvia species, collected from different regions of Turkey, were prepared. The phenolic contents of these extracts, found to be 7 phenolic acids (caffeic, chlorogenic, p-coumaric, salicylic, syringic, rosmarinic and t-ferulic acids), 8 flavonoids (apigenin, isorhamnetin, kaempferol, luteolin, penduletin, quercetin-3,6-dimethylether, quercetin and salvigenin), 5 flavonoid glycosides (isoquercetin, kaempferol-3-rutinoside, luteolin-7-O-glycoside, quercitrin and rutin hydrate), pyrogallol, vanillin and fumaric acid were quantitatively determined by LC-MS/MS method. In Salvia species, in general, caffeic acid (7.11-185.03 μg/g), fumaric acid (8.32-2119.85 μg/g), luteolin (12.92-344.68 μg/g), penduletin (35-740.57 μg/g), rosmarinic acid (134.6-4420 μg/g) and rutin hydrate (5.23-7522.31 μg/g) were detected. The salvinorin contents of these extracts was also determined by LC-MS/MS method. While salvinorin A was found to be the most abundant in S. recognita, S. cryptantha and S. glutinosa (212.86 ± 20.46 μg/g, 51.50 ± 4.95 μg/g and 38.92 ± 3.74 μg/g), salvinorin B was found to be rich in S. potentillifolia, S. adenoculon and S. cryptantha (2351.99±232.22 μg/g, 768.78±75.90 μg/g and 402.24±39.71 μg/g), respectively. The volatile organic compounds of the plants were determined by thermal desorption gas chromatography mass spectrometry method. Although the contents of the volatile organic compounds differed among the species, mainly borneol (0.09-37.4%), β-caryophyllene (0.23-10.27%), spathulenol (0.03-14.07%), caryophyllene oxide (0.03-11.25%), β-bisabolon (0.21-7.96%), bicyclohexane (0.06-33.31%), 1,8-cineole (eucalyptol) (0.04-23.1%) and camphor (0.05-13%) were determined. For genomic DNA analysis, the 5S rRNA coding gene sequences of the plant species were elucidated, and similarities and universal kinship between species were identified. The DNA sequences of the Salvia plants and the DNA sequence of the Salvia divinorum plant from GenBank were aligned with the multiple sequence alignment programs and the similarities of the species to the hallucinogenic Salvia divinorum plant were revealed. It has been determined that the DNA sequences of Salvia species usually are between 300 bp and 500 bp. But Salvia nemorosa (467 bp), Salvia glutinosa (466 bp), Salvia russellii (424 bp) and Salvia verticillata (269 bp) were, in general, found to differ from other Salvia species by 300 bp base length. In the literature, the Salvia divinorum plant was reported to have a base length of 487 bp. The base sequences of the species were arranged by Clusteal W multiple sequence alignment program. Thus, similarities to the Salvia divinorum plant were investigated chemometrically and genomically. The total phenolic and flavonoid contents of the extracts were determined to be equivalent to pyrocatechol and quercetin, using Folin-Ciocalteu and aluminum nitrate method, respectively. The total phenolic and the total flavonoid contents of the extracts were found to be between 13.90±0.56-164.06±2.82 μg/mg and 14.15±0.11-58.57±0.00 μg/mg, respectively. ABTS cation radical scavenging activity and Copper (II) ion antioxidant capacity (CUPRAC) were determined to be equivalent to Trolox. Radical scavenging capacity toward ABST cation and CUPRAC activity capacity were determined to be between 0.16±0.00-1.55±0.00 mmol TR/g and 0.25±0.00-2.47±0.00 mmol TR/g, respectively. DPPH free radical scavenging and lipid peroxidation inhibition (β-carotene-linoleic acid assay) were studied at 4 different concentrations (10, 25, 50 and 100 μg/mL) in % inhibition. The results were concluded comparing with α-tocopherol, BHT and BHA standards. Antimicrobial activities were examined, applying disc diffusion method, against Gram negative bacteria E. coli, Gram positive bacteria S. aureus, yeast C. albicans and mycobacteria M. smegmatis. As standard, negative control DMSO, positive control Gentamycin, Oxacillin, Ketoconazole and Streptomycin antibiotics were used. While Salvia blepharochlaena and Salvia modesta species demonstrated activity toward M. smegmatis, Salvia kronenburgii and Salvia poculata species showed activity against S. aureus. Moreover, toward S. aureus, Salvia heldreichiana good, Salvia aucheri, Salvia hypargeia, Salvia modesta and Salvia trichoclada modest, Salvia bracteata, Salvia euphratica, Salvia huberi, Salvia glutinosa and Salvia nemorosa little antimicrobial activities they showed.