Influence of the Abiotic Elicitors Ag Salts of Aspartic Acid Derivatives, Self-Organized in Nanofibers with Monomeric and Dimeric Molecular Structures, on the Antioxidant Activity and Stevioside Content in Micropropagated Stevia rebaudiana Bert

Abstract

The use of nanomaterials in biotechnology for the in vitro propagation of medical plants and the accumulation of certain biologically active metabolites is becoming an efficient strategy. This study aimed to evaluate the influence of the concentration (0, 1, 10, 50, and 100 mg L^-1) of two types of nanofibers on the growth characteristics, the antioxidant status, and the production of steviol glycosides in micropropagated Stevia rebaudiana Bert. plantlets. The nanofibers were synthesized by aspartic acid derivatives (L-Asp) Ag salts self-organized into nanofibers with two different molecular structures: monomeric, containing one residue of L-Asp with one hydrophilic head which bonds one Ag ion (NF1-Ag salt); and dimeric, containing two residues of L-Asp with two hydrophilic heads which bond two Ag ions (NF2-Ag salt). An increase in the shoots from the explants' number and length, biomass accumulation, and micropropagation rate was achieved in the plants treated with the NF1-Ag salt in concentrations from 1 to 50 mg L^-1 after 30 days of in vitro proliferation compared to the NF2-Ag salt. In contrast, the plants grown on MS media supplemented with NF2-Ag salt exhibited an increase in the level of stevioside, rebaudioside A, and mono- (CQA) and dicaffeoylquinic (DCQA) acids as compared to the NF1-Ag salt.

Keywords: Stevia rebaudiana Bert.; antioxidant activity; dicaffeoylquinic acids (DCQA); in vitro propagation; monocaffeoylquinic acids (CQA); steviol glycosides.

Figure 8

Organic compounds used for the preparation of silver salts: (a) dekanoyl-L-Asp-N-hexylamide and (b) N,N′-Bis(N-dekanoyl-L-Aspartyl)-diaminohexane.

Figure 1

The activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (GPX) in S. rebaudiana plantlets in vitro propagated on MS media I, and on MS media supplemented with various concentrations (1, 10, 50, 100 mg L⁻¹) of nanofibers synthesized by the derivatives of the L-aspartic acid with a monomeric (NF1-Ag salt) or dimeric molecular structure (NF2-Ag salt) carrier of silver ions. 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” or “A” for the lowest data value and ascending to the next letters for higher data value. The statistical analysis of NF1-Ag salt (uppercase) and NF2-Ag salt (lowercase) was performed separately.

Figure 2

The content of metabolites with antioxidant power (total phenolic compounds (A) and flavonoids (B), WS-AOM (C) and LS-AOM (D)) and antioxidant potential (DPPH (E), FRAP (F)) in S. rebaudiana plantlets in vitro propagated on MS media, and on MS media supplemented with various concentrations (1, 10, 50, 100 mg L⁻¹) of nanofibers synthesized by the derivatives of the L-aspartic acid with a monomeric (NF1-Ag salt) or dimeric molecular structure (NF2-Ag salt) carrier of silver ions. 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” or “A” for the lowest data value and ascending to the next letters for higher-data value. The statistical analysis of NF1-Ag salt (uppercase) and NF2-Ag salt (lowercase) was performed separately.

Figure 3

The levels of stress markers (MDA, H₂O₂, proline, and -SH groups) in the S. rebaudiana plantlets in vitro propagated on MS media, and on MS media supplemented with various concentrations (1, 10, 50, 100 mg L⁻¹) of nanofibers synthesized by the derivatives of the L-aspartic acid with a monomeric (NF1-Ag salt) or dimeric molecular structure (NF2-Ag salt) carrier of silver ions. 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” or “A” for the lowest data value and ascending to the next letters for higher-data value. The statistical analysis of NF1-Ag salt (uppercase) and NF2-Ag salt (lowercase) was performed separately.

Figure 4

Stevioside, rebaudioside A, and sugar content in the S. rebaudiana plantlets in vitro propagated on MS media free of NP, and on MS media supplemented with various concentrations (1, 10, 50, 100 mg L⁻¹) of nanofibers synthesized by the derivatives of the L-aspartic acid with a monomeric (NF1-Ag salt) or dimeric molecular structure (NF2-Ag salt) carrier of silver ions. Values are means ± SE, n = 6; different letters indicate significant differences assessed by the Fisher LSD test (p ≤ 0.05) after performing ANOVA one-way analysis. We use the letter “a” for the lowest data value and ascend to the next letters for higher data value.

Figure 5

Content of total mono- (CQA) and dicaffeoylquinic (DCQA) acids (mg/g DW) and CQA/D CQA ratio in the S. rebaudiana plantlets in vitro propagated on MS media free of NP, and on MS media supplemented with various concentrations (1, 10, 50, 100 mg L⁻¹) of nanofibers synthesized by the derivatives of the L-aspartic acid with a monomeric (NF1-Ag salt) or dimeric molecular structure (NF2-Ag salt) carrier of silver ions. Values are means ± SE, n = 6; different letters indicate significant differences assessed by the Fisher LSD test (p ≤ 0.05) after performing ANOVA one-way analysis. We use the letter “a” for the lowest data value and ascend to the next letters for higher data value.

Figure 6

Biplot from PCA performed on the mean content of the individual compounds in different extracts of Stevia rebaudiana plantlets in vitro propagated on MS media free of NP, and on MS media supplemented with nanofibers synthesized by the derivatives of the L-aspartic acid with a monomeric (NF1-Ag salt) or dimeric molecular structure (NF2-Ag salt) carrier of silver ions.

Figure 7

Molecular structures of the silver-organic salts used in our experiments: (a) NF1-Ag salt and (b) NF2-Ag salt.