Unveiling the interaction profile of rosmarinic acid and its bioactive substructures with serum albumin

Abstract

Rosmarinic acid, a phytochemical compound, bears diverse pharmaceutical profile. It is composed by two building blocks: caffeic acid and a salvianic acid unit. The interaction profile, responsible for the delivery of rosmarinic acid and its two substructure components by serum albumin remains unexplored. To unveil this, we established a novel low-cost and efficient method to produce salvianic acid from the parent compound. To probe the interaction profile of rosmarinic acid and its two substructure constituents with the different serum albumin binding sites we utilised fluorescence spectroscopy and competitive saturation transfer difference NMR experiments. These studies were complemented with transfer NOESY NMR experiments. The thermodynamics of the binding profile of rosmarinic acid and its substructures were addressed using isothermal titration calorimetry. In silico docking studies, driven by the experimental data, have been used to deliver further atomic details on the binding mode of rosmarinic acid and its structural components.

Keywords: Rosmarinic acid; STD-NMR; caffeic acid; salvianic acid; serum albumin.

Scheme 1.

Structures of the studied compounds: rosmarinic acid, caffeic acid, salvianic acid, ibuprofen and warfarin.

Figure 1.

Hydrolysis of rosmarinic acid.

Figure 2.

Fluorescence spectra of the interaction of BSA (2 μΜ) with (a) rosmarinic acid (A–Χ: 0–20 μΜ), (b) caffeic acid (A–Χ: 0–20 μΜ) and (c) salvianic acid (A–Χ: 0–20 μΜ).

Figure 3.

(a) ¹H NMR reference spectrum of the complex rosmarinic acid (2 mM) – BSA (20 μM) in Tris-d₁₁ buffer 10 mM, pH = 7.4 with 600 μL D₂O. (b) STD difference NMR spectrum of the complex rosmarinic acid–BSA. The percentages values show the STD_AMP for all the protons of rosmarinic acid.

Figure 4.

Relative STD_AMP (saturation transfer difference amplification factor) values of the saturated protons (regarding their binding to BSA) of rosmarinic acid (black) and its submoieties, caffeic acid (red) and salvianic acid (blue). The values in each molecule are normalised to the intensity of the signal with the largest STD effect.

Figure 5.

(a) Expanded region of tr-NOESY spectrum of the complex rosmarinic acid–BSA, recorded with mixing time of 350 ms at 600 MHz, showing the presence of negative NOEs of bound ligand. (b) Expanded region of tr-NOESY spectrum of the complex caffeic acid–salvianic acid–BSA, recorded with mixing time of 700 ms at 800 MHz showing the presence of negative NOEs, which are related to the protons of caffeic acid (labelled in red) and presence of positive NOEs, which are related to the protons of the major stereoisomer of salvianic acid (labelled in blue). For the minor isomer of salvianic acid only trivial NOEs between the H2 and H3 protons, which are overlaid with the zero-order artefacts of scalar coupling, are present.

Figure 6.

(a) ¹H NMR reference spectrum of the complex rosmarinic acid (2 mM) – BSA (50 μΜ), including warfarin 2 mM, in PBS buffer 10 mM, pH = 7.4 with 600 μL D₂O. STD difference NMR spectrum of the complex rosmarinic acid–BSA, including: (b) 2 mM warfarin. (c) 4 mM warfarin (d) 6 mM warfarin. (e) 8 mM warfarin (details for the protons of rosmarinic acid in Figure 3 and for warfarin in Supplementary Figure S13).

Figure 7.

(a) ¹H NMR reference spectrum of the complex caffeic acid (2 mM) – BSA (50 μΜ), including warfarin 2 mM, in PBS buffer 10 mM, pH = 7.4 with 600 μL D₂O. STD difference NMR spectrum of the complex caffeic acid–BSA, including: (b) 2 mM warfarin, (c) 4 mM warfarin, (d) 6 mM warfarin, (e) 8 mM warfarin (details for the protons of caffeic acid in Supplementary Figure S5 and for warfarin in Supplementary Figure S13).

Figure 8.

(a) ¹H NMR reference spectrum of the complex salvianic acid (2 mM) – BSA (50 μΜ), including warfarin 2 mM, in PBS buffer 10 mM, pH = 7.4 with 600 μL D₂O. STD difference NMR spectrum of the complex salvianic acid–BSA, including: (b) 2 mM warfarin, (c) 4 mM warfarin (details for the protons of salvianic acid in Supplementary Figure S6 and for warfarin in Supplementary Figure S13).

Figure 9.

(a) ¹H NMR reference spectrum of the complex rosmarinic acid–warfarin–BSA (0.2 mM:2 mM:20 μΜ), in Tris buffer 10 mM, pH = 7.4. (b) STD difference spectrum of the complex rosmarinic acid–warfarin–BSA (0.2 mM:2 mM:20 μΜ), in Tris buffer 10 mM, pH = 7.4. (c) ¹H NMR reference spectrum of the complex salvianic acid–warfarin–BSA (0.2 mM:2 mM:20 μΜ), in Tris buffer 10 mM, pH = 7.4 (d) STD difference spectrum of the complex salvianic acid–warfarin–BSA (0.2 mM:2 mM:20 μΜ), in Tris buffer 10 mM, pH = 7.4 (details for the protons of rosmarinic acid in Figure 3, for salvianic acid in Supplementary Figure S6 and for warfarin in Supplementary Figure S13).

Figure 10.

(a) ¹H NMR reference spectrum of the complex rosmarinic acid (2 mM) BSA (50 μΜ), including ibuprofen 2 mM, in PBS buffer 10 mM, pH = 7.4 with 600 μL D₂O. STD difference NMR spectrum of the complex rosmarinic acid–BSA, including: (b) 2 mM ibuprofen (c) 4 mM ibuprofen (details for the protons of rosmarinic acid in Figure 3 and for ibuprofen in Supplementary Figure S14).

Figure 11.

(a) ¹H NMR reference spectrum of the complex caffeic acid (2 mM) – BSA (50 μΜ), including ibuprofen 2 mM, in PBS buffer 10 mM, pH = 7.4 with 600 μL D₂O. STD difference NMR spectrum of the complex caffeic acid–BSA, including: (b) 2 mM ibuprofen (c) 4 mM ibuprofen (details for the protons of caffeic acid in Supplementary Figure S5 and for ibuprofen in Supplementary Figure S14).

Figure 12.

(a) ¹H NMR reference spectrum of the complex salvianic acid (2 mM) – BSA (50 μΜ), including ibuprofen 2 mM, in PBS buffer 10 mM, pH = 7.4 with 600 μL D₂O. STD difference NMR spectrum of the complex salvianic acid–BSA, including: (b) 2 mM ibuprofen, (c) 4 mM ibuprofen (details for the protons of salvianic acid in Supplementary Figure S6 and for ibuprofen in Supplementary Figure S14).

Figure 13.

(a) ¹H NMR reference spectrum of the complex ibuprofen (2 mM) BSA (20 μΜ), including salvianic acid 0.2 mM, in Tris-d₁₁ buffer 10 mM, pH = 7.4 with 600 μL D₂O. STD difference NMR spectrum of the complex ibuprofen–BSA, including: (b) 0.2 mM salvianic acid, (c) 3 mM salvianic acid (details for the protons of salvianic acid in Supplementary Figure S6 and for ibuprofen in Supplementary Figure S14).

Figure 14.

Isothermal titration calorimetry measurements for the interaction of BSA with (a) Rosmarinic acid and (b) Caffeic acid. For each interaction the isotherm plot (up) and the fitting of the integrated curve (down) are being presented.

Figure 15.

Best pose of rosmarinic acid in Sudlow site I. The nine favourable hydrogen bonds and π-π stacking between Arg256 and aromatic ring A (Supplementary Figure S17) can explain its highly favourable binding.

Figure 16.

Binding interactions of caffeic acid in Sudlow site I of BSA.

Figure 17.

Best pose of BSA–salvianic acid in Sudlow site I. The four favourable hydrogen bonds, the salt bridge between Arg198–carboxylic anion and the π–cation can explain its highly favourable binding.