Flavonoid Profile of the Genista tridentata L., a Species Used Traditionally to Treat Inflammatory Processes

Abstract

Ethnopharmacological surveys on Portuguese flora reveal that Genista tridentata L. is a shrub used in traditional medicine for the treatment of various inflammation-related health problems, although scientific support of its benefits is still necessary. In order to establish the anti-inflammatory potential of G. tridentata and support its traditional use, ethanolic extracts of three sections of the plant (root, stem, and leaves) were subjected to in vitro evaluation of anti-inflammatory activity using lipopolysaccharide (LPS)-stimulates macrophages as an inflammation model. Simultaneously, we also aimed to establish the extracts' flavonoids profile. The ethanolic extracts, obtained by Soxhlet extraction, profile of the three sections confirmed their richness in flavonoids, being three prenylated flavonoids isolated and characterized in the root, including a new natural compound, the 3-methoxymundulin. The extracts from the three plant sections showed strong antioxidant activity at the cellular level and significantly inhibit the LPS-triggered NO production by downregulating Nos2 gene transcription and consequently iNOS expression. Additionally, root and stem extracts also decreased the LPS-induced transcription of the pro-inflammatory genes Il1b, Il6, and Ptgs2. Thus, the results support the anti-inflammatory properties attributed to G. tridentate preparations. Relevantly, the roots of the shrub, plant part not used, is an unexplored source of compounds with pharmacological and nutraceutical value.

Keywords: 3-methoxymundulin; Genista tridentata; UHPLC-DAD-ESI/MSn profile; anti-inflammatory activity; flavonoids; lupinifolin; mundulin.

Figure 1

Effect of G. tridentata extracts on Raw 264.7 macrophages viability. Cells were exposed to different concentrations of the extracts, during 24 h and then viability was assessed by the resazurin assay. Data is presented as percentage of untreated cells (control) and represent the mean from three independent experiments.

Figure 2

Evaluation of the capacity of G. tridentata extracts to modulate NO production in Raw 264.7 macrophages. (a) Levels of NO in supernatants from cells stimulated with extracts or with extracts and LPS. (b) Assessment of the potential of NO scavenging activity through the SNAP assays. Data is presented as concentration of NO and represent mean ± SD from three independent biological experiments. (**** p < 0.0001: control vs. LPS; #### p < 0.0001: LPS vs. extracts + LPS). Data is presented as percentage of control (nontreated cells/medium) and represent the mean ± SD from at least three independent experiments.

Figure 3

Effects of G. tridentata extracts on the LPS-triggered production of ROS by macrophages. Cells were cultured in the indicated conditions and the ROS production was assessed with H₂DCFDA (green), a ROS-sensitive fluorescent probe. Hoechst (blue) was used to label the nuclei. Images representative of different fields were acquired at a magnification of 63 × (scale bar = 20 µm). Data is presented as mean fluorescence intensity ± SD from three independent experiments. (**** p < 0.0001: C vs. LPS; #### p < 0.0001: LPS vs. extracts + LPS).

Figure 4

Effects of G. tridentata extracts over LPS-induced transcription of Nos2, Ptgs2, Il1b, Il6, and Tnfa genes. Cells were exposed to extracts from root, stem, and leaves and then stimulated with LPS. After 24 h the mRNA was extracted and the gene transcription was assessed by q-PCR. Data is presented as mRNA fold change relatively to untreated cells (C) and represent the mean ± SD from three independent experiments. (* p < 0.05; ** p < 0.01: C vs. LPS; # p < 0.05; ## p < 0.01; #### p < 0.0001: LPS vs. extracts + LPS).

Figure 5

Impact of G. tridentata extracts on the LPS-induced expression of iNOS and COX-2. Cells were exposed to extracts from root, stem, and leaves and then stimulated with LPS. After 24 h total cell lysates were prepared and iNOS and COX-2 protein levels were assessed by Western blot. The results are expressed as percentage of optical densities relative to untreated cells (control). Equal protein loading was controlled using antibody against β-tubulin. A representative blot is shown and each value on graphs represents the mean ± SD of three independent experiments. (**** p < 0.0001: C vs. LPS; # p < 0.05, #### p < 0.0001: LPS vs. extracts + LPS).

Figure 6

UHPLC chromatograms of G. tridentata extracts. (A) Stems extract (S) recorded at 230 nm; (B) leaves extract (L) recorded at 240 nm; (C) roots extract (R) recorded at 280 nm.

Figure 6

UHPLC chromatograms of G. tridentata extracts. (A) Stems extract (S) recorded at 230 nm; (B) leaves extract (L) recorded at 240 nm; (C) roots extract (R) recorded at 280 nm.

Figure 7

Isoflavonoid derivatives identified in G. tridentata extracts.

Figure 8

Other polyphenolic derivatives identified in G. tridentata extracts.

Figure 9

Prenylated flavonoids isolated from G. tridentata roots extract.

Figure 10

Representative types of flavonoids present in G. tridentata extracts.