Antioxidant Capacity and Accumulation of Caffeoylquinic Acids in Arnica montana L. In Vitro Shoots After Elicitation with Yeast Extract or Salicylic Acid

Abstract

Arnica montana L. is an important herbal medicinal plant that belongs to the family Asteraceae. This plant has been known for its medicinal uses for centuries. A. montana exhibits several pharmacological properties, including immunomodulatory, anti-inflammatory, anticancer, antioxidant, and antibacterial effects. For the first time, the impacts of the biotic elicitor yeast extract, and the abiotic elicitor salicylic acid on micropropagation, antioxidant potential, and accumulation of caffeoylquinic acids in arnica in vitro shoots were assessed. The results showed that yeast extract applied at 100 mg/L significantly promotes shoot multiplication, biomass yield, total phenolic content, and synthesis of caffeoylquinic acids compared to control untreated shoots. Flavonoid content was the highest in samples treated with 200 mg/L of yeast extract, although at this concentration the measured biometric parameters began to decrease. Salicylic acid at 100 µM was found to be effective in the induction of vigorous shoots, shoot height growth, and biomass accumulation; nevertheless, this elicitor downregulated the caffeoylquinic acid level, total phenolics, and flavonoids. Increasing the concentration of salicylic acid to 200 µM caused shoot multiplication and fresh biomass accumulation reduction. Both elicitors modulated the activity of antioxidant enzymes against oxidative stress. Overall, the use of these substances can improve the growth and biomass yield in Arnica in vitro shoots.

Keywords: HPLC; antioxidant potential; caffeoylquinic acids; micropropagation; total flavonoid content; total phenolic content.

Figure 1

Shoot culture of Arnica montana L. after 5 weeks cultivation on MS medium containing 0.5 mg/L BAP and different concentrations of yeast extract (A) control without YE, (B) 50 mg/L YE, (C) 100 mg/L YE and (D) 200 mg/L YE. The scale bar represents: 1 cm.

Figure 2

The activity of antioxidant enzymes superoxide dismutase (SOD) (A), catalase (CAT) (B), ascorbate peroxidase (APX) (C), and guaiacol peroxidase (GPX) (D) in Arnica montana shoots elicited with YE applied at different concentrations (0, 50, 100 and 200 mg/L). Values are means ± SE, n = 20; different letters indicate significant differences assessed by the Fisher LSD test (p ≤ 0.05) after performing ANOVA one-way analysis. We used the letter “a” for the highest data value and descended to the next for lower data values.

Figure 3

The content of metabolites with antioxidant power (TPC (A) and TFC (B), WS-AOM (C), and LS-AOM (D)) in Arnica montana shoots elicited with YE applied at different concentrations (0, 50, 100, and 200 mg/L). Values are means ± SE, n = 20; different letters indicate significant differences assessed by the Fisher LSD test (p ≤ 0.05) after performing ANOVA one-way analysis. We used the letter “a” for the highest data value and descended to the next for lower data values.

Figure 4

Antioxidant potential—DPPH free radical scavenging activity (A) and ferric-reducing antioxidant power (FRAP) (B) in Arnica montana shoots elicited with YE applied at different concentrations (0, 50, 100, and 200 mg/L). Values are means ± SE, n = 20; different letters indicate significant differences assessed by the Fisher LSD test (p ≤ 0.05) after performing ANOVA one-way analysis. We used the letter “a” for the highest data value and descended to the next for lower data values.

Figure 5

UHPLC-MS base peak chromatogram of A. montana shoot extract in negative mode.

Figure 6

HPLC chromatogram at 320 nm of a standard mixture of CQAs, control, and sample treated with 100 mg/L YE.

Figure 7

Shoot culture of Arnica montana L. after 5 weeks of cultivation on MS medium containing 0.5 mg/L BAP and different concentrations of salicylic acid (A) control without SA, (B) 50 µM SA, (C) 100 µM SA, and (D) 200 µM SA. The scale bar represents: 1 cm.

Figure 8

The activity of antioxidant enzymes superoxide dismutase (SOD) (A), catalase (CAT) (B), ascorbate peroxidase (APX) (C), and guaiacol peroxidase (GPX) (D) in Arnica montana shoots elicited with SA applied at different concentrations (0, 50, 100 and 200 µM). Values are means ± SE, n = 20; different letters indicate significant differences assessed by the Fisher LSD test (p ≤ 0.05) after performing ANOVA one-way analysis. We used the letter “a” for the highest data value and descended to the next for lower data values.

Figure 9

The content of metabolites with antioxidant power (TPC (A) and TFC (B), WS-AOM (C), and LS-AOM (D)) in Arnica montana shoots elicited with SA applied at different concentrations (0, 50, 100, and 200 µM). Values are means ± SE, n = 20; different letters indicate significant differences assessed by the Fisher LSD test (p ≤ 0.05) after performing ANOVA one-way analysis. We used the letter “a” for the highest data value and descended to the next for lower data values.

Figure 10

Antioxidant potential—DPPH free radical scavenging activity (A) and ferric-reducing antioxidant power (FRAP) (B) in Arnica montana shoots elicited with SA applied at different concentrations (0, 50, 100 and 200 µM). Values are means ± SE, n = 20; different letters indicate significant differences assessed by the Fisher LSD test (p ≤ 0.05) after performing ANOVA one-way analysis. We used the letter “a” for the highest data value and descending to the next for lower data values.

Figure 11

HPLC chromatogram at 320 nm of a standard mixture of CQAs, control, and sample treated with 100 µM SA.