Origanum majorana Extracts: A Preliminary Comparative Study on Phytochemical Profiles and Bioactive Properties of Valuable Fraction and By-Product

Abstract

Origanum majorana L. (O. majorana) (Lamiaceae) is an aromatic Mediterranean plant widely used in food, cosmetics, and traditional medicine due to its aroma and rich content of bioactive compounds. While its leaves and flowers are commonly utilized, lignified stems are often discarded. This study compared hydroalcoholic extracts from the leaves and flowers (valuable fraction, VF) and stems (by-product, BP). Phytochemical analysis revealed qualitatively similar profiles, identifying 20 phenolic compounds, with Rosmarinic acid and Salvianolic acid B as the most and second most abundant, respectively. Antioxidant activity was evaluated in vitro using DPPH (IC₅₀ [µg/mL]: VF 30.11 ± 3.46; BP 31.72 ± 1.46), H₂O₂ (IC₅₀ [µg/mL]: VF 103.09 ± 4.97; BP 119.55 ± 10.58), and O₂^•- (IC₅₀ [µg/mL]: VF 0.71 ± 0.062; BP 0.79 ± 0.070). Both extracts (20 µg/mL) fully restored oxidative balance in hemin-stressed AC16 cardiomyocytes, without altering the expression of catalase, heme-oxygenase 1, superoxide dismutase 2, or ferritin. Anti-inflammatory activity in LPS-stimulated RAW 264.7 macrophages showed that VF (IC₅₀ 400 µg/mL) reduced ^•NO release to control levels, while BP achieved a ~60% reduction. Cytotoxicity was assessed on cancer cell lines: CaCo-2 (IC₅₀ [µg/mL]: VF 154.1 ± 6.22; BP 305.2 ± 15.94), MCF-7 (IC₅₀ [µg/mL]: VF 624.6 ± 10.27; BP 917.9 ± 9.87), and A549 (IC₅₀ [µg/mL]: VF 720.8 ± 13.66; BP 920.2 ± 16.79), with no cytotoxicity on normal fibroblasts HFF-1 (IC₅₀ > 1000 µg/mL for both extracts). Finally, both extracts slightly inhibited only CYP1A2 (IC₅₀ [µg/mL]: VF 497.45 ± 9.64; BP 719.72 ± 11.37) and CYP2D6 (IC₅₀ [µg/mL]: VF 637.15 ± 14.78, BP 588.70 ± 11.01). These results support the potential reuse of O. majorana stems as a sustainable source of bioactive compounds for nutraceutical and health-related applications.

Keywords: Rosmarinic acid; cancer; cytochrome P450 isoforms; inflammation; oxidative stress; polyphenols; sustainability.

Figure 1

Bombus terrestris on O. majorana L. (Lamiaceae) flowers at the collection site. Syracuse, Italy, June 2022.

Figure 2

HPLC/DAD chromatograms, visualized at 330 nm of VF (black line) and BP (blue line) extracts from O. majorana. See text for further information and experimental details.

Figure 3

Effect of VF and BP hydroalcoholic extracts on cell viability in A549, CaCo-2, and MCF-7 cancer cell lines and HFF-1 normal cells. MTT tests were performed on the various cell lines treated with different concentrations of extract (from 50 to 1000 μg/mL) for 72 h. Data are represented as the means ± S.D. of three independent experiments. Confidence intervals were calculated by one-way ANOVA test: * Significant vs. untreated control cells; ^† BP significant vs. VF at the same concentration; p < 0.05.

Figure 4

Cytotoxic effect of VF and BP hydroalcoholic extracts from O. majorana in AC16 cardiomyocytes. MTT tests were performed in AC16 cells treated with different concentrations of extract (from 20 to 200 μg/mL) for 48 h. Data are represented as the means ± S.D. of three independent experiments. Confidence intervals were calculated by one-way ANOVA test.

Figure 5

Cytotoxic effect of VF and BP hydroalcoholic extracts from O. majorana in RAW 264.7 macrophages. MTT tests were performed in RAW 264.7 cells treated with different concentrations of extract (from 50 to 400 μg/mL) for 24 h. Data are represented as the means ± S.D. of three independent experiments. Confidence intervals were calculated by one-way ANOVA test.

Figure 6

Antioxidant activity of VF or BP extracts from O. majorana in AC16 cardiomyocytes challenged with hemin. Untreated AC16 cells (CTRL) and cells pretreated with different concentrations of extracts were exposed to hemin (H, 1 µM). Each result represents the mean ± S.D. of three experiments. Confidence intervals were calculated by one-way ANOVA test. * Significant vs. non-stressed control cells; # significant vs. hemin-stressed cells; ^† BP significant vs. VF at the same concentration; p < 0.05.

Figure 7

Gene expression levels of antioxidant genes in AC16 cardiomyocytes after exposure to the VP and BP extracts. qPCR analysis of relative gene expression of four endogenous antioxidant enzymes: heme oxygenase-1 (HMOX-1), ferritin heavy chain 1 (FTH1), mitochondrial superoxide dismutase (SOD2), and catalase (CAT) in untreated AC16 (CTRL), and cells pretreated with different concentrations of VF or BP extracts from O. majorana. Each result represents the mean ± S.D. of three experiments. Confidence intervals were calculated by one-way ANOVA test.

Figure 8

^•NO released in RAW 264.7 macrophages challenged with LPS in the presence of VF or BP extracts from O. majorana. Untreated RAW 264.7 cells (CTRL), cells pretreated with different concentrations of VF or BP extracts, and/or cells activated with LPS (2 µg/mL) were used in these experiments. Each result represents the mean ± S.D. of four experiments. Confidence intervals were calculated by one-way ANOVA test. * Significant vs. non-stressed control cells; # significant vs. LPS-activated cells; ^† BP significant vs. VF at the same concentration; p < 0.05.