Chemical profiling and in vitro biological properties of extracts containing rosmarinic acid, obtained from Thymus sp. and Rosmarinus sp. (Lamiaceae)
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Title of the project: “Chemical profiling and in vitro biological properties of extracts containing rosmarinic acid, obtained from Thymus sp. and Rosmarinus sp. (Lamiaceae)”
Project acronym: RosThym
Project code: PN-III-P1-1.1-PD-2019-0967
Contract number: PD 207 - 28/08/2020
Approved budget: 246,950 RON
Project leader: Lecturer Irina IELCIU, PhD
Mentor: Professor ILIOARA ELENA ONIGA, PhD
Department of Pharmacognosy, „Iuliu Haţieganu” University of Medicine and Pharmacy, 12 Ion Creangă, 3rd floor, 400010, Cluj-Napoca, Romania
BrainMap ID: U-1700-038K-8191
Research project description:
Thymus marshallianus Willd. and Rosmarinus officinalis L. (Lamiaceae family) are two species known for their content of volatile oils and polyphenolic compounds. Among the most important polyphenolic compounds in the composition of these species, rosmarinic acid (RA), a compound with remarkable biological activities, such as anti-inflammatory, antimicrobial or antidiabetic ones, can be found. At the basis of these activities the antioxidant mechanism is found, the oxidative processes being responsible for numerous effects at various levels in these pathologies. In this context, the present project aimed to bring important arguments to support the protective activity of AR and the extracts of the two species against glycemic stress, involved in the pathology of diabetes mellitus (DM) and its complications.
In stage 1 of the project, the collection and processing of plant material belonging to the two species was carried out and plant extracts were prepared and analyzed by HPLC/MS, for the identification and quantification of the main polyphenols, including RA. Phytochemical analysis of these extracts highlighted increased amounts of RA for both species. In the composition of the extract obtained from the leaves of R. officinalis, phenolic terpenes were identified, while in the composition of the aerial parts of the T. marchallianus species, compounds from the flavonoid class were identified.
In stage 2 of the project, extracts enriched in polyphenolic compounds were prepared and subjected to comparative HPLC-DAD analysis for the identification and quantification of polyphenolic compounds before and after enrichment. Phytochemical analysis of these extracts highlighted the presence of polyphenols in significantly increased amounts after enrichment. Subsequently, these extracts were studied to highlight their biological activities. In the case of the T. marschallianus species, a comparative study of two types of extracts, obtained from specimens collected from the spontaneous flora and from experimental cultures, was carried out. Effects on human umbilical vein endothelial cells (HUVEC) exposed to normoglycemic and hyperglycemic conditions were studied. Effects on hyperglycemia were assessed by quantifying oxidative stress and NF-ĸB, pNF-ĸB, HIF-1α and γ-H2AX expressions. Polyphenol-enriched extracts showed a significant reduction in oxidative stress and γ-H2AX formation and enhanced HIF-1α expression suggesting their protective role on endothelial cells in hyperglycemia. The tested extracts reduced the total expression of NF-κB and diminished its activation under hyperglycemic conditions. In the case of the R. officinalis species, the evaluation of the biological activities involved the in vitro study of an extract enriched in polyphenols on the viability of human hepatocellular carcinoma cells (HepG2) and on three bacterial strains, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli strains. Cytotoxicity against HepG2 cells was evaluated against two normal cell lines: oral tissue-derived mesenchymal stromal cells (pMSC) and the human foreskin fibroblast cell line (Hs27). The extracts showed cytotoxic properties on HepG2 cells and did not affect the viability of normal cells. Antimicrobial activity was also significant, showing the most intense effect on Gram-positive species.
In stage 3 of the project, the optimization and validation of a specific analytical method for the analysis of polyphenols from the composition of the two species was carried out and the protective biological activity against the oxidative processes induced by diabetes in experimental animals on the heart and aorta of diabetic rats was studied. The effects of the administration of the extracts belonging to the two species and RA on the aortic rings were thus analyzed, in order to evaluate the responses in the contractile processes stimulated by phenylephrine and relaxation to acetylcholine, together with parameters such as malondialdehyde (MDA), superoxide dismutase (SOD), nitric oxide synthase 3 (NOS 3), activated AMP protein kinase (AMPK), adiponectin and oxidized glutathione (GSSG) in hearts and aortas of diabetic rats. The results showed that the administration of extracts and RA alleviated oxidative stress by decreasing the level of MDA, GSSG, SOD, thus proving antioxidant properties in the oxidative processes induced by DM in the heart and aorta. For the R. officinalis species, the incorporation into liposomes was carried out, in the intent of formulating specific pharmaceutical forms, which were characterized, optimized and tested for the antiproliferative and antimicrobial effects. The loading of the obtained liposomes was analyzed using the tested and validated HPLC/MS method and the biological assays for the evaluation of the antiproliferative activity consisted of the evaluation of cell viability on a liver adenocarcinoma cell line and a human liver cell line, while the antimicrobial assays were performed by the diffusimetric method and by microdilution tests. The obtained liposomes showed high encapsulation efficiency, suitable particle size and good stability. Polyphenols were identified in their composition in important quantities, and cell viability and apoptosis tests showed an intense effect on cancerous cell lines and a totally different pattern on normal cells, indicating a selective toxicity towards cancerous ones and an antiproliferative mechanism. Antimicrobial potential evaluation tests revealed an effect on all tested bacteria, with better efficacy against Gram-positive species.
All the obtained results provide important scientific arguments for the possibility of using extracts from these species in the adjuvant treatment of DM.