Phytochemical Composition and Cytoprotective Properties of the Endemic Sideritis sipylea Boiss Greek Species: A Valorization Study

Abstract

Sideritis sipylea Boiss. (Fam. Lamiaceae) is an endemic plant of the North Aegean Islands (Greece), commonly known as ironwort. Traditionally, its aerial parts have been used to relieve several ailments, especially gastrointestinal disorders, however, with scant knowledge about the pharmacological basis. In the present study, an endemic S. sipylea Greek species from Lesvos Island has been characterized for phytochemical composition and biological activities, in order to give a possible scientific basis to its traditional use and to highlight a further nutraceutical interest as a source of bioactive phytochemicals and extracts. Three different fractions obtained from a methanolic extract of S. sipylea aerial parts by using ethyl acetate with 10 (S10), 20 (S20), and 50% (S50) methanol as fractionation solvents were phytochemically characterized. Moreover, their antioxidant power and cytoprotective activity in different human cell lines were evaluated. The phytochemical analysis highlighted the presence of flavonoids, iridoids, and phenolic acids in all the tested samples. Particularly, the S10 fraction mainly contained iridoids, while S20 and S50 lavandulifolioside and chlorogenic acid, respectively. The fractions also showed antioxidant properties, S10 and S20 being the most potent. When assessed in human cholangiocytes, they counteracted the cytotoxicity of the tBOOH pro-oxidant agent, by reducing ROS levels and affecting GSH antioxidant system. The present findings highlight a possible interest in S10 and S20 fractions from S. sipylea as sources of bioactive molecules and stimulate further studies in order to characterize their possible application for nutraceutical and pharmaceutical purposes.

Keywords: Greek medicinal plants; North Aegean; UPLC-HRMS/MS; advanced glycation end products; antioxidant activity; antiproliferative activity; chelating activity; mountain tea; oxidative stress; phenolics.

Figure 1

Scavenger activity towards (A) DPPH, (B) ABTS, and (C) NO radicals of S10, S20, and S50 fractions from Sideritis sipylea Boiss. Data represent the average and standard error of at least three independent experiments (n = 3).

Figure 2

Ability of S10, S20, and S50 fractions from Sideritis sipylea Boiss to chelate ferrous (A) and ferric (B) ions, and to reduce ferric (C) ones. Data represent the average and standard error of at least three independent experiments (n = 3).

Figure 3

Cytotoxicity of S10, S20, and S50 fractions from Sideritis sipylea Boiss in nonmalignant mouse macrophages RAW264.7 (A–C), human bronchial epithelium BEAS-2B cells (D–F), and human intrahepatic cholangiocytes H69 (G–I) determined by MTT assay after 24 h of exposure. Data displayed as mean ± SE of at least three independent experiments (n = 3). * p < 0.05 and *** p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s multiple comparison post-test).

Figure 4

Cytotoxicity of S10, S20, and S50 fractions from Sideritis sipylea Boiss in nonmalignant mouse macrophages RAW264.7 (A–C), human bronchial epithelium BEAS-2B cells (D–F), and human intrahepatic cholangiocytes H69 (G–I) determined by Neutral Red assay after 24 h exposure. Data displayed as mean ± SE of at least three technical replicates from two experiments (n = 3). ** p < 0.01 and *** p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s multiple comparison post-test).

Figure 5

Cytoprotective activity of the positive control quercetin (Q) and of S10, S20, and S50 fractions from Sideritis sipylea Boiss towards the tBOOH-induced oxidative damage in nonmalignant mouse macrophages RAW264.7 (A–C), human bronchial epithelium BEAS-2B cells (D–F), and human intrahepatic cholangiocytes H69 (G–I) determined by MTT assay. Data displayed as mean ± SE of at least three technical replicates from two experiments (n = 3). °°° p < 0.001 vs. control (Student’s t-test); * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. tBOOH (one-way ANOVA followed by Dunnett’s multiple comparison post-test).

Figure 6

Cytoprotective activity of the positive control quercetin (Q) and S10, S20, and S50 fractions from Sideritis sipylea Boiss towards the tBOOH-induced oxidative damage in nonmalignant mouse macrophages RAW264.7 (A–C), human bronchial epithelium BEAS-2B cells (D–F), and human intrahepatic cholangiocytes H69 (G–I) determined by Neutral Red assay. Data displayed as mean ± SE of at least three technical replicates from two experiments (n = 3). °°° p < 0.001 vs. control (Student’s t-test); * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. tBOOH (one-way ANOVA followed by Dunnett’s multiple comparison post-test).

Figure 7

Effect of S10 (A), S20 (B), and S50 (C) fractions from Sideritis sipylea Boiss on the ROS levels induced by the pro-oxidant agent tBOOH in H69 cells. ROS levels are expressed as oxidation index with respect to the basal levels. Data are mean ± SE from at least three independent experiments (n = 3). ° p < 0.05, °° p < 0.01, and °°° p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s multiple comparison post-test); * p < 0.05 and *** p < 0.001 vs. tBOOH (one-way ANOVA followed by Dunnett’s multiple comparison post-test).

Figure 8

Representative images of intracellular ROS after treatment of H69 normal cholangiocytes with tBOOH (500 µM) and S10, S20, and S50 fractions (100 µg/mL) alone or in combination with the pro-oxidant agent. Original magnification 10X.

Figure 9

Effect of S10, S20, and S50 fractions from Sideritis sipylea Boiss on the GSH/GSSG ratio after treatment with the pro-oxidant agent tBOOH in H69 cells. GSH and GSSH were evaluated in cell lysates and calculated with respect to the calibration curves of GSH and GSSG. Data are mean ± SE from at least three independent experiments (n = 3). ° p < 0.05 and °°° p < 0.001 vs. control (one-way ANOVA followed by Dunnett’s multiple comparison post-test); *** p < 0.001 vs. tBOOH (one-way ANOVA followed by Dunnett’s multiple comparison post-test).