Comparison of the Phytochemical Variation of Non-Volatile Metabolites within Mother Tinctures of Arnica montana Prepared from Fresh and Dried Whole Plant Using UHPLC-HRMS Fingerprinting and Chemometric Analysis

Abstract

Arnica montana L. has been recognized for centuries as an herbal remedy to treat wounds and promote healing. It also has a long tradition of use in homeopathy. Depending on its medicinal utilization, standardization regulations allow different manufacturing processes, implying different raw materials, such as the whole arnica plant in its fresh or dried state. In this study, an untargeted metabolomics approach with UHPLC-HRMS/MS was used to cross-compare the phytochemical composition of mother tinctures of A. montana that were prepared from either fresh whole plant (fMT) matter or from oven-dried whole plant (dMT) matter. The multivariate data analysis showed significant differences between fMT and dMT. The dereplication of the HRMS and MS/MS spectra of the more discriminant compounds led to annotated quinic acid, dicaffeoyl quinic acids, ethyl caffeate, thymol derivatives and dehydrophytosphingosine, which were increased in fMT, while Amadori rearrangement products (ARP) and methoxyoxaloyl-dicaffeoyl quinic acid esters were enhanced in dMT. Neither sesquiterpene lactones nor flavonoids were affected by the drying process. This is the first time that a sphingosine, ethyl caffeate and ARP are described in A. montana. Moreover, putative new natural products were detected as 10-hydroxy-8,9-epoxy-thymolisobutyrate and an oxidized proline fructose conjugate, for which isolation and full structure elucidation will be necessary to verify this finding.

Keywords: Arnica montana; dried vs. fresh materials; metabolomics; mother tinctures; phytochemical differences.

Figure 1

PCA score plot of the first two principal components (PC1 and PC2), based on the LC-MS dataset, including 924 compounds. Mother tinctures from the fresh plants (F), mother tinctures from dried plants (D) and quality control (QC).

Figure 2

Orthogonal partial least squares discriminant analysis (OPLS-DA) between the mother tinctures prepared from fresh (F) and dried (D) whole plants of Arnica montana in positive- and negative-ion ESI-MS. (A) OPLS-DA score plot, (B) OPLS-DA loading S-plot.

Figure 3

Ionic current chromatogram of the m/z 251.15 positive ion (fMT) corresponding to the proposed 10-hydroxy-8,9-epoxy-thymolisobutyrate (above) and the MS/MS spectrum of the m/z 251.15 ion, with proposed structures for fragment ions (below). The position of the positive charge is arbitrary when several oxygens are present.

Figure 4

Negative-ion MS/MS spectrum of the [M-H]⁻ parent trihydroxy thymol at m/z 197, with proposed structures for the product ions.

Figure 5

Proposed structures for compounds increased in the mother tinctures, prepared from fresh whole plants of Arnica montana.

Figure 6

Proposed structures for the compounds increased in mother tinctures prepared from the dried whole plant of Arnica montana.

Figure 7

MS/MS spectrum of the [M-H]⁻ ion at m/z 278, showing the structures of the diagnostic product ions, mainly due to the characteristic cleavages occurring in the hexose moiety. These product ions from glycoconjugates are denoted according to the nomenclature introduced by Domon and Costello [43].