Coumarins

The calculations were carried out by employing the Gaussian09 software package [24 ]. For optimization of neutral molecules, the corresponding radicals, anions, and radical cations quantum chemical calculations based on density functional theory (DFT) were used. Since it has been shown that the M062-X method well describes short-range and medium-range intramolecular and intermolecular interactions (<500 pm) [25 ], the M06-2X method is applied in combination with 6–311++G(d,p) basis set. This theoretical model is suitable for thermodynamic and kinetic investigation of reaction mechanisms of examined compounds with free radicals [22 (link),26 (link)]. All investigated structures were reoptimized in methanol (ε = 32.61), and benzene (ε = 2.27). The solvent effect was included by the Conductor-like Polarizable Continuum Model (CPCM) [27 (link)], without any geometrical constraints. Solvents were selected to simulate the polar and non-polar environments, as well as conditions of experimental measurements.
Three antioxidant mechanisms were selected for the evaluation of the antioxidant activity: Hydrogen Atom Transfer (HAT), Single-Electron Transfer followed by Proton Transfer (SET-PT), and Sequential Proton Loss-Electron Transfer (SPLET) [28 (link),29 (link),30 (link),31 (link)].
The abstraction of hydrogen atoms can be described by two mechanisms, HAT and Proton-Coupled Electron Transfer (PCET) [29 (link)]. The HAT mechanism is defined as the simultaneous transfer of an electron and a proton between the donor and acceptor. On the other hand, the PCET mechanism involves a significant redistribution of molecular charge, during the transfer of hydrogen atoms. In this study, only the HAT mechanism was considered because only thermodynamic parameters were used to test antioxidant capacity. It should be noted that both reaction pathways are necessary for the kinetic examination of the mechanisms of antioxidant activity. In the HAT mechanism, the homolytic cleavage of the O-H bond occurs leading to the separation between radical species (Ar-O^•) and a hydrogen atom (Equation (1)) [30 (link),31 (link)].

The SET-PT mechanism is a two-step process that includes electron loss from a molecule and generation of the radical cation (Ar–OH^+•), which in the second step loses protons (Equation (2a,b)).


The SPLET mechanism is also a two-step mechanism. In the first step, an anion (Ar–O⁻) is formed from the antioxidant molecule, while the corresponding radical is formed after the electron transfer in the second step of this mechanism (Equation (3a,b)).


The thermodynamic parameters governing the mentioned antioxidative mechanisms are BDE (Bond Dissociation Enthalpy) describing HAT, PA (Proton Affinity), and ETE (Electron Transfer Enthalpy) describing SPLET, and IP (Ionization Potential) and PDE (Proton Dissociation Enthalpy) describing SET-PT mechanism. The contribution of these parameters was calculated from the enthalpies of the optimized chemical species using the following equations [32 (link),33 (link)]:




All reaction enthalpies used in the equations were calculated at 298 K. The calculated enthalpy values of the solvated proton, H(H⁺) and electron, H(e⁻), for the M062-X method in benzene (−856.9 and –11.9 kJ moL⁻¹) and methanol (−1010.5 and −93.5 kJ moL⁻¹) were taken from the literature [28 (link),34 (link)].
The free radical scavenging potency of the synthesized coumarin derivatives toward the DPPH radical was investigated in two different solvents: benzene and methanol. Mechanisms of radical scavenging activity are described by Equations (1s)–(5s), given in the Supplementary Material. Thermodynamic parameters of the investigated mechanisms were calculated according to Equations (6s)–(10s) from Supplementary material.

UV-Vis spectrophotometrical assays were used to determine the total content of flavonoids (as mg/g quercetin equivalents) [23 (link)], catechins (as mg/g (+)-catechin equivalents) [24 (link)], procyanidins (as mg/g procyanidin B₁ equivalents) [25 (link)], phenylpropanoids (as mg/g rosavin equivalents) [26 (link)], gallotannins (as mg/g gallic acid equivalents) [27 (link)], ellagitannins (as mg/g ellagic acid equivalents) [28 (link)], coumarins (as mg/g umbelliferon equivalents) [29 (link)], and anthocyanes (as mg/g cyanidin-3-O-glucoside equivalents) [30 (link)] in dry herbal samples of R. rosea (roots, rhizomes, leaves, flowers, stems). All the analyses were carried out in triplicate and the data were expressed as mean value ± standard deviation (SD).
Antioxidant activity of total extracts and selected compounds was determined using spectrophotometric assays. Trolox was used as a positive control (PC; 10 mg/mL), and water was used as a negative control (NC). Scavenging activity against 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH^•) was studies as the following assay: 500 μL DPPH^• (freshly prepared MeOH solution, 100 μg/mL) and 500 μL of Rhodiola rosea extract (freshly prepared 50% MeOH solution, 1–200 μg/mL) or pure compound (freshly prepared MeOH solution, 1–200 μg/mL). Absorbance (520 nm) was measured after 15 min. The DPPH^• scavenging capacity was calculated using equation: Scavenging capacity (%) = ((A₅₂₀^NC – A₅₂₀^PC) – (A₅₂₀^Sample – A₅₂₀^PC)/(A₅₂₀^NC – A₅₂₀^PC)) × 100, where A₅₂₀^NC is the absorbance of the negative control, A₅₂₀^PC is the absorbance of the positive control, and A₅₂₀^Sample is the absorbance of the sample solution. For studing 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radicals (ABTS^•+) scavenging capacity ABTS (water solution; 7 mM) reacted with potassium persulphate (water solution; final concentration 2.45 mM) in the dark at 20 °C (12–16 h before use). The ABTS^•+ solution was diluted with MeOH to an absorbance of 0.70 at 734 nm and equilibrated at 20 °C. Rhodiola rosea extract (500 μL; freshly prepared 50% MeOH solution, 1–200 μg/mL) was mixed with ABTS^•+ solution (500 μL) and the absorbance was measured at 734 nm after 20 min. The ABTS^•+ scavenging capacity was calculated using equation: Scavenging capacity (%) = ((A₇₃₄^NC – A₇₃₄^PC) – (A₇₃₄^Sample – A₇₃₄^PC)/(A₇₃₄^NC – A₇₃₄^PC)) × 100, where A₇₃₄^NC is the absorbance of the negative control, A₇₃₄^PC is the absorbance of the positive control, and A₇₃₄^Sample is the absorbance of the sample solution. Superoxide radicals (O₂^•−) scavenging capacity was determined using Rhodiola rosea extract (50 μL; freshly prepared solution in Tris-HCl buffer, 0.05 M, pH 8.2; 10–1000 μg/mL) mixed with pyrogallol (50 μL, 6 mM) and Tris-HCl buffer (1 mL). The absorbance was measured at 325 nm after 5 min. The O₂^•− scavenging capacity was calculated using equation: Scavenging capacity (%) = ((A₃₂₅^NC – A₃₂₅^PC) – (A₃₂₅^Sample – A₃₂₅^PC)/(A₃₂₅^NC – A₃₂₅^PC)) × 100, where A₃₂₅^NC is the absorbance of the negative control, A₃₂₅^PC is the absorbance of the positive control, and A₃₂₅^Sample is the absorbance of the sample solution. To determine hydroxyl radicals (^•OH) scavenging capacity Rhodiola rosea extract (100 μL; freshly prepared solution in 0.2 M phosphate buffer (pH 7.4; 1–500 μg/mL) mixed with deoxyribose solution in the same buffer (100 μL; 2.8 mM), H₂O₂ (10 μL; 3.6 mM), FeCl₃ (10 μL; 5.0 mM) and EDTANa₂ (100 μL; 100 μM). After addition of ascorbic acid (50 μL; 200 μM) the mixture was incubated at 55 °C for 20 min. Finally, 2-thiobarbituric acid (800 μL; 10 mg/mL) and trichloroacetic acid (800 μL; 50 mg/mL) were added and heated at 95 °C for 20 min. The absorbance was measured at 530 nm. The ^•OH scavenging capacity was calculated using equation: Scavenging capacity (%) = ((A₅₃₀^NC – A₅₃₀^PC) – (A₅₃₀^Sample – A₅₃₀^PC)/(A₅₃₀^NC – A₅₃₀^PC)) × 100, where A₅₃₀^NC is the absorbance of the negative control, A₅₃₀^PC is the absorbance of the positive control, and A₅₃₀^Sample is the absorbance of the sample solution. The IC₅₀ value is the effective concentration at which free radicals (DPPH^•, ABTS^•+, O₂^•−, ^•OH) was scavenged by 50%. Values are expressed as mean obtained from five independent experiments. Carotene bleaching assay was performed as described previously using β-carotene as a substrate (Sigma-Aldrich, St. Louis, MO, USA, cat. No. C9750) [31 (link)].

To 1.5 mL of each C. sulphureus extract, 1.5 mL of 10% NaOH was added. A color change to yellow indicated the presence of coumarins.