Phytochemical and pharmacological characterization of primula species from Georgian Flora

Although primula species are abundant in phytochemicals, little is known about their pharmacological characteristics, especially those of endemic species. The purpose of this study was to examine the biological activity and phytochemical composition of the Georgian endemic Primula saguramica,as well as Primula macrocalyx, and the Caucasian endemic Primula woronowii. Folin–Ciocalteu and aluminum chloride colorimetric assays were used to measure the total phenolic content (TPC) and total flavonoid content (TFC), respectively. Cytotoxicity was assessed using the MTT assay in RAW 264.7 murine macrophages (ATCC-TIB-71), A549 lung adenocarcinoma cell line (ATCC-CCL-185), U87MG human glioblastoma cell line (ATCC-HTB-14), and NIH/3T3 - murine fibrobalst cell line (ATCC-CRL-1658),  in addition to the hemolysis assay on human erythrocytes. The ABTS radical scavenging assay was used to confirm the antioxidant activity. An individual compound from the most active fraction (P.w.3, 50% methanolic fraction of P. woronowii) was isolated and identified using preparative high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). For the quantitative determination of the individual compound in crude extracts and fractions, HPLC analysis was carried out, and its predicted pharmacological profile was evaluated using an in silico approach. P.w.3 had the highest TPC and TFC content (41.84 ± 0.08 mg GAE/g and 36.02 ± 0.2 mg QE/g) among all studied samples. It did not exhibit cytotoxic or hemolytic activity; however, it revealed potent antioxidant capacity (IC₅₀ = 7.35 ± 0.23 µg/mL), which was significantly stronger than clitorin (13.25 ± 0.24 µg/mL) and the crude extract P.w.1 (42.01 ± 0.21 µg/mL). A flavonoid glycoside, clitorin (kaempferol 3-O-(2'',6''-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside), was identified from P.w.3 Clitorin content in P.w.3 was approximately ~5-fold higher (16.93 ± 1.16%, RSD) compared with P.w.1 (2.86 ± 2.5%, RSD). An in silico analysis showed that clitorin may have multifunctional biological activities, such as antioxidant, anti-inflammatory, antiproliferative, and vasoprotective effects. P.w.3 contained abundant bioactive flavonoids and exhibited strong antioxidant properties, without displaying cytotoxic or hemolytic effects. The isolated compound, clitorin, may serve as a lead in the discovery of phytotherapeutics for oxidative stress associated diseases.