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. 2020 Jun 3;9(6):482.
doi: 10.3390/antiox9060482.

Thymus zygis subsp. zygis an Endemic Portuguese Plant: Phytochemical Profiling, Antioxidant, Anti-Proliferative and Anti-Inflammatory Activities

Amélia M Silva  1   2 , Carlos Martins-Gomes  2   3 , Eliana B Souto  4   5 , Judith Schäfer  6 , João A Santos  2   7 , Mirko Bunzel  6 , Fernando M Nunes  3   8
Affiliations

Thymus zygis subsp. zygis an Endemic Portuguese Plant: Phytochemical Profiling, Antioxidant, Anti-Proliferative and Anti-Inflammatory Activities

Amélia M Silva et al. Antioxidants (Basel). .

Abstract

Thymus zygis subsp. zygis is an endemic Portuguese plant belonging to the Thymus zygis species. Although T. zygis is commonly used as a condiment and as a medicinal herb, a detailed description of the polyphenol composition of hydroethanolic (HE) and aqueous decoction (AD) extracts is not available. In this work, we describe for the first time a detailed phenolic composition of Thymus zygis subsp. zygis HE and AD extracts, together with their antioxidant, anti-proliferative and anti-inflammatory activities. Unlike other Thymus species, T. zygis subsp. zygis extracts contain higher amounts of luteolin-(?)-O-hexoside. However, the major phenolic compound is rosmarinic acid, and high amounts of salvianolic acids K and I were also detected. T. zygis subsp. zygis extracts exhibited significant scavenging activity of ABTS+, hydroxyl (•OH), and nitric oxide (NO) radicals. Regarding the anti-proliferative/cytotoxic effect, tested against Caco-2 and HepG2 cells, the AD extract only slightly reduced cell viability at higher concentrations (IC50 > 600 µg/mL, 48 h exposure), denoting very low toxicity, while the HE extract showed a high anti-proliferative effect, especially at 48 h exposure (IC50 of 85.01 ± 15.10 μg/mL and 82.19 ± 2.46 μg/mL, for Caco-2 and HepG2, respectively). At non-cytotoxic concentrations, both extracts reduced the nitric oxide (NO) release by lipopolysaccharide (LPS)-stimulated RAW 264.7 cells (at 50 μg/mL, HE and AD extracts inhibited NO release in ~89% and 48%, respectively). In conclusion, the results highlight the non-toxic effect of aqueous extracts, both resembling the consumption of antioxidants in foodstuff or in functional food. Furthermore, the HE extract of T. zygis subsp. zygis is a source of promising molecules with antioxidant, anti-inflammatory and anticancer activities, highlighting its potential as a source of bioactive ingredients for nutraceutical and pharmaceutical industries.

Keywords: Thymus zygis subsp. zygis; anti-inflammatory activity; anti-proliferative activity; antioxidant; aqueous decoction; hydroethanolic extract; luteolin-O-hexoside; phenolic profiling; radical scavenging activity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Phenolic profile of hydroethanolic (HE) (A) and aqueous decoction (AD) (B) extracts of Thymus zygis subsp. zygis, obtained by High-performance liquid chromatography, coupled to diode array detector (HPLC-DAD). For peak number identification, please refer to Table 2.
Figure 2
Figure 2
Chromatogram of oleanolic acid (OA) and ursolic acid (UA) standards and of Thymus zygis hydroethanolic (HE) extract.
Figure 3
Figure 3
Anti-proliferative effect of Thymus zygis subsp. zygis aqueous decoction (AD) and hydroethanolic (HE) extracts on Caco-2 (A and B for AD and HE extracts, respectively) and HepG2 cells (C and D for AD and HE extracts, respectively). Two exposure times, 24 and 48 h, were considered, as indicated. Results are expressed as (mean ± SD, n = 4). Statistically significant differences (p < 0.05) between the control and sample concentrations at respective incubation time are denoted by *, and those between exposure periods at the same concentration are denoted by #.
Figure 4
Figure 4
Anti-inflammatory activity of Thymus zygis subsp. zygis extracts. (A) Inhibition of nitric oxide (NO) release by LPS-stimulated RAW 264.7 cells induced by aqueous decoction (AD; left bars, red) and by hydroethanolic (HE; right bars, blue) extracts, expressed as percentage of control (see methods for details). (B) Effect of AD (red bars) and HE (blue bars) extracts on RAW 264.7 cells viability (see methods for details). Results are expressed as mean ± SD (n = 4 independent assays).
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