Novel Signposts on the Road from Natural Sources to Pharmaceutical Applications: A Combinative Approach between LC-DAD-MS and Offline LC-NMR for the Biochemical Characterization of Two Hypericum Species (H. montbretii and H. origanifolium)

Abstract

The members of the genus Hypericum have great potential to develop functional uses in nutraceutical and pharmaceutical applications. With this in mind, we aimed to determine the chemical profiling and biological properties of different extracts (ethyl acetate, methanol and water) from two Hypericum species (H. montbretii and H. origanifolium). We combined two approaches (LC-DAD-MS and LC-NMR) to identify and quantify chemical compounds of the extracts. Antioxidant properties (free radical quenching, reducing power and metal chelating) and enzyme inhibitory effects (cholinesterase, tyrosinase, amylase and glucosidase) were determined as biological properties. The tested extracts were rich in caffeic acid derivatives and flavonoids, and among them, 3-caffeoyl quinic acid and myricetin-3-O-rhamnoside were found to be the main compounds. The total phenolic and flavonoid levels were determined to be 50.97-134.99 mg GAE/g and 9.87-82.63 mg RE/g, respectively. With the exception of metal chelating, the methanol and water extracts showed stronger antioxidant properties than the ethyl acetate extracts. However, different results were obtained for each enzyme inhibition assay, and in general, the ethyl acetate extracts present more enzyme-inhibiting properties than the water or methanol extracts. Results from chemical and biological analyses were combined using multivariate analysis, which allowed establishing relationships between composition and observed effects of the Hypericum extracts based on the extraction solvents. To gain more insights between chemical compounds and enzyme-inhibiting effects, we performed molecular docking analysis. We observed favorable interactions between certain compounds and the tested enzymes during our analysis, confirming the data obtained from the multivariate approach. In conclusion, the obtained results may shed light on the road from natural sources to functional applications, and the tested Hypericum species may be considered potential raw materials, with promising chemical constituents and biological activities.

Keywords: Hypericum; bioactive agents; flavonoids; hydroxycinnamic acids; natural enzyme inhibitors.

Figure 1

Workflow of the different measurements described in the paper.

Figure 2

Superimposition of the ¹H-NMR spectra of Hypericum montbretii ethyl acetate fractions (A–D). Blue square indicate the signals of phenolic compounds (T indicates signals due to toluene from the chromatographic fractionation process).

Figure 3

Structures of main compounds detected in the H. montbretii and origanifolium.

Figure 4

Enlarged part of the ¹H-NMR spectra of Hypericum montbretii ethyl acetate fractions (C in red and D in blue). For assignments, see text.

Figure 5

Superimposition of the ¹H-NMR spectra of Hypericum montbretii ethyl acetate fractions. C (blue spectrum) and D (red spectrum) signals ascribable to quinic acid moieties (K) and sugar residue are indicated.

Figure 6

H-NMR of H. origanifolium water fraction obtained from sephadex.

Figure 7

Exemplificative chromatograms at 330 nm of H. montbretii and H. origanifolium extracts.

Figure 8

Binding energy (docking) scores of the bioactive compounds from Hypericum extracts.

Figure 9

Protein-ligand interaction: (A) AChE and quercetin 3-O-rhamnoside isomer 1, (B) BChE and 3-O-rhamnoside isomer 1, (C) tyrosinase and myricetin 3-O-rhamnoside, (D) amylase and quercetin 3-O-galactoside (hyperoside) and (E) glucosidase and 3-caffeoylquinic acid.

Figure 10

PLS-DA obtained using the matrix obtained with all the quantitative data for the different solvents of each Hypericum specie and the results of bioassays. Three replicate were used for generating the plot. Solvents are indicated as EA ethyl acetate, MEOH methanol, Water, number indicate the replicates.

Figure 11

Loading scatter plot corresponding to plot 8, showing the correlation between the compounds that have been detected in the extracts (green dots) and the considered bioassays. Blue dots represent Acetyl Cholinesterase AChea, Butyril Cholinesterase BuCea, Glucosidase GLCa, Tyrosinase TYRa, Amylase AMYa, Total phenolic contents TPC, CUPRAC, FRAP, DPPH, ABTS and Phosphomolibdenum PHA.

Figure 12

Biplot showing the loading scatter plot of the model generated using the quantitative data on the chemical composition of the extracts and the results of bioassays. Plant extracts are represented with blue squares, enzyme inhibitory test are represented with red dots, metal chelating and ferric reducing power are represented with brown dots, antioxidant assay results are represented with turquoise dots and compounds are all represented with green dots.