Extraction of Anthraquinones from Japanese Knotweed Rhizomes and Their Analyses by High Performance Thin-Layer Chromatography and Mass Spectrometry.

Vesna Glavnik,Irena Vovk

doi:10.3390/plants9121753

Abstract

Anthraquinones (yellow dyes) were extracted from Japanese knotweed rhizomes with twelve extraction solvents (water; ethanol(aq) (20%, 40%, 60%, 70% and 80%), ethanol, 70% methanol(aq), methanol, 70% acetone(aq), acetone and dichloromethane). The obtained sample test solutions (STSs) were analyzed using high-performance thin-layer chromatography (HPTLC) coupled to densitometry and mass spectrometry (HPTLC–MS/MS) on HPTLC silica gel plates. Identical qualitative densitometric profiles (with anthraquinone aglycones and glycosylated anthraquinones) were obtained for STSs in all the solvents except for the STS in dichloromethane, which enabled the most selective extractions of anthraquinone aglycones emodin and physcion. The highest extraction efficiency, evaluated by comparison of the total peak areas in the densitograms of all STSs scanned at 442 nm, was achieved for 70% acetone(aq). In STS prepared with 70% acetone(aq), the separation of non-glycosylated and glycosylated anthraquinones was achieved with developing solvents toluene–acetone–formic acid (6:6:1, 3:6:1 and 3:3:1 v/v) and dichloromethane–acetone–formic acid (1:1:0.1, v/v). Non-glycosylated anthraquinones were separated only with toluene–acetone–formic acid, among which the best resolution between emodin and physcion gave the ratio 6:6:1 (v/v). This solvent and dichloromethane–acetone–formic acid (1:1:0.1, v/v) enabled the best separation of glycosylated anthraquinones. Four HPTLC-MS/MS methods enabled the identification of emodin and tentative identification of its three glycosylated analogs (emodin-8-O-hexoside, emodin-O-acetyl-hexoside and emodin-O-malonyl-hexoside), while only the HPTLC-MS/MS method with toluene-acetone-formic acid (6:6:1, v/v) enabled the identification of physcion. Changes of the shapes and the absorption maxima (bathochromic shifts) in the absorption spectra after post-chromatographic derivatization provided additional proof for the detection of physcion and rejection of the presence of chrysophanol in STS.

Highlights

Japanese knotweed (Fallopia japonica Houtt., Polygonaceae; synonyms: Polygonum cuspidatum, Polygonum reynoutria and Reynoutria japonica) is on the list of the “100 World’s Worst Invasive AlienSpecies”, because it represents huge ecological and economic problems in Europe and North America
sample test solutions (STSs) prepared with 70% ethanol(aq), ethanol, 70% methanol(aq), methanol, 70% acetone(aq), acetone and dichloromethane were on the first plate together with standards emodin and aloin A (Figure 1)
The obtained sample test solutions (STSs) were analyzed by the high-performance thin-layer chromatography (HPTLC) method on HPTLC silica gel plates developed with toluene–acetone–formic acid (3:6:1, v/v)

Summary

Introduction

Japanese knotweed (Fallopia japonica Houtt., Polygonaceae; synonyms: Polygonum cuspidatum, Polygonum reynoutria and Reynoutria japonica) is on the list of the “100 World’s Worst Invasive AlienSpecies”, because it represents huge ecological (biodiversity loss) and economic problems (damage of infrastructure) in Europe and North America. Japanese knotweed (Fallopia japonica Houtt., Polygonaceae; synonyms: Polygonum cuspidatum, Polygonum reynoutria and Reynoutria japonica) is on the list of the “100 World’s Worst Invasive Alien. In the environment of its origin, which is Eastern Asia, it is used in traditional Chinese and Japanese medicine for healing infections, inflammatory diseases, hyperlipidemia and other diseases [1]. Plants 2020, 9, 1753 infusions) are usually prepared from rhizomes (subterranean stems). Leaves of Japanese knotweed rhizomes have been shown to be rich in proanthocyanidins [15]. Anthraquinones are the largest group of natural dyes, with about 700 compounds [17], and can be found in many plant genera, such as Cassia [18,19], Aloe [17], Rheum [20] and Fallopia [2], to which Rhizomes contain many secondary metabolites, including stilbenes (especially trans-resveratrol and its glycosylated analogs [2,3,4,5,6,7,8,9,10,11]); proanthocyanidins (from monomers [2,6,8,12,13], dimers [2,6,8,12,13], oligomers [2,4,8,12,13] and up to polymers [2,8,12,13]); phenolic acids [2,6,8]; phenylpropanoid glycosides [2,4]; flavonoids [2,8]; naphthalenes [2,3,4,6]; triterpenoids [8] and anthraquinones (especially emodin [2,3,5,6,7,9,10,11,14], physcion [2,3,5,6,7,9,10,11,14] and their glycosides [2,3,4,6,9,11,14] and other anthraquinones [2,4,6,7,12]).

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