Flavan-3-ols and Proanthocyanidins in Japanese, Bohemian and Giant Knotweed

Abstract

Flavan-3-ols and proanthocyanidins of invasive alien plants Japanese knotweed (Fallopia japonica Houtt.), giant knotweed (Fallopia sachalinensis F. Schmidt) and Bohemian knotweed (Fallopia × bohemica (Chrtek & Chrtkova) J.P. Bailey) were investigated using high performance thin-layer chromatography (HPTLC) coupled to densitometry, image analysis and mass spectrometry (HPTLC-MS/MS). (+)-Catechin, (-)-epicatechin, (-)-epicatechin gallate and procyanidin B2 were found in rhizomes of these three species, and for the first time in Bohemian knotweed. (-)-Epicatechin gallate, procyanidin B1, procyanidin B2 and procyanidin C1 were found in giant knotweed rhizomes for the first time. Rhizomes of Bohemian and giant knotweed have the same chemical profiles of proanthocyanidins with respect to the degree of polymerization and with respect to gallates. Japanese and Bohemian knotweed have equal chromatographic fingerprint profiles with the additional peak not present in giant knotweed. Within the individual species giant knotweed rhizomes and leaves have the most similar fingerprints, while the fingerprints of Japanese and Bohemian knotweed rhizomes have additional peaks not found in leaves. Rhizomes of all three species proved to be a rich source of proanthocyanidins, with the highest content in Japanese and the lowest in Bohemian knotweed (based on the total peak areas). The contents of monomers in Japanese, Bohemian and giant knotweed rhizomes were 2.99 kg/t of dry mass (DM), 1.52 kg/t DM, 2.36 kg/t DM, respectively, while the contents of dimers were 2.81 kg/t DM, 1.09 kg/t DM, 2.17 kg/t DM, respectively. All B-type proanthocyanidins from monomers to decamers (monomers-flavan-3-ols, dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers and decamers) and some of their gallates (monomer gallates, dimer gallates, dimer digallates, trimer gallates, tetramer gallates, pentamer gallates and hexamer gallates) were identified in rhizomes of Bohemian knotweed and giant knotweed. Pentamer gallates, hexamers, hexamer gallates, nonamers and decamers were identified for the first time in this study in Bohemian and giant knotweed rhizomes.

Keywords: HPTLC–MS; Polygonaceae; Polygonum; Reynoutria; catechins; chemical profiling; condensed tannins; fingerprints; flavanols; procyanidins.

Figure 1

HPTLC chromatograms for the qualitative determination of flavan-3-ols and proanthocyanidins in STSs from rhizomes (2 μL, 50 mg/mL) of Japanese (track 6), Bohemian (track 7) and giant (track 8) knotweed based on standards. The HPTLC silica gel plate was developed with tol uene—acetone—formic acid (3:6:1, v/v) and documented at white light after derivatization with DMACA detection reagent. Applications of standards: (−)-gallocatechin gallate (0.2 µg; track 1), (−)-catechin gallate (0.2 µg; track 2), procyanidin C1 (0.3 µg; track 3), procyanidin B3 (0.2 µg; track 4), (−)-epicatechin (0.1 µg; track 5, higher R_F), (−)-epigallocatechin (0.2 µg; track 5, lower R_F), (+)-catechin (0.1 µg; track 9, higher R_F) (−)-gallocatechin (0.2 µg; track 9, lower R_F), (−)-epicatechin gallate (0.2 µg; track 10), procyanidin B1 (0.2 µg; track 11), procyanidin B2 (0.2 µg; track 12) (−)-epigallocatechin gallate (0.2 µg; track 13).

Figure 2

HPTLC chromatograms for the qualitative determination of flavan-3-ols and proanthocyanidins in STSs from rhizomes (1 μL, 50 mg/mL) of Japanese (track 6), Bohemian (track 7) and giant (track 8) knotweed based on standards. The HPTLC cellulose plates were developed with water (A), 1-propanol–water–acetic acid (4:2:1, v/v) (B), 1-propanol–water–acetic acid (20:80:1, v/v) (C), and documented at white light after derivatization with DMACA detection reagent. The ap plications of standards: (−)-gallocatechin gallate (60 ng; track 1), (−)-catechin gallate (60 ng; track 2), procyanidin C1 (150 ng; track 3), procyanidin B3 (100 ng; track 4), (−)-epicatechin (50 ng; track 5, higher R_F), (−)-epigallocatechin (60 ng; track 5, lower R_F), (+)-catechin (50 ng; track 9, higher R_F), (−)-gallocatechin (60 ng; track 9, lower R_F), (−)-epicatechin gallate (60 ng; track 10), procyanidin B1 (120 ng; track 11), procyanidin B2 (90 ng, track 12) (−)-epigallocatechin gallate (60 ng; track 13).

Figure 3

HPTLC chromatograms used for the quantitative determination of proanthocyanidins in STSs from rhizomes (1 μL, 50 mg/mL) of Japanese (tracks 2 and 8), Bohemian (tracks 4 and 10) and giant (tracks 6 and 12) knotweed and standard solutions of (−)-epicatechin and procyanidin B2. The HPTLC silica gel plate was developed with toluene–acetone–formic acid (3:6:1, v/v) and documented at white light after derivatization with DMACA detection reagent. The applications of (−)-epicatechin and procyanidin B2 standard solutions: track 1: 30 ng; track 3: 40 ng; track 5: 60 ng; track 7: 80 ng; track 9: 100 ng; track 11: 120 ng; track 13: 150 ng.

Figure 4

The densitograms of STSs (1 μL, 50 mg/mL) from rhizomes of Japanese (JK), giant (GK) and Bohemian (BK) knotweed and standard solutions (STD, 40 ng) of (−)-epicatechin (EC) and procyanidin B2 (B2) scanned in absorption/reflectance mode at 280 nm before the derivatization (A) and at 655 nm after the derivatization with DMACA reagent (B). The HPTLC silica gel plate was developed with toluene–acetone–formic acid (3:6:1, v/v).

Figure 5

Comparisons of the videodensitogram of standards (−)-epicatechin (EC; R_F = 0.82) and procyanidin B2 (B2; R_F = 0.63) (30 ng; dashed green line) with the videodensitograms of the fingerprint profiles of STSs (1 μL, 50 mg/mL) from rhizomes of Japanese (black line), Bohemian (blue line) and giant knotweed (red line). The videodensitograms were obtained in absorption mode by image analysis of the HPTLC silica gel plate after the development with toluene–acetone–formic acid (3:6:1, v/v) and after the derivatization with DMACA detection reagent. The asterisk (*) indicates the peaks that are specific to Japanese and Bohemian knotweed rhizomes.

Figure 6

Comparisons of the videodensitogram fingerprint profiles of STSs (1 μL, 50 mg/mL) from leaves and rhizomes of the same knotweed species (Japanese (A), Bohemian (B) and giant (C) knotweed) with the videodensitogram of standards (−)-epicatechin (EC; R_F = 0.82) and procyanidin B2 (B2; R_F = 0.63) (30 ng; dashed green line). The videodensitograms were obtained in absorption mode by image analysis of the HPTLC silica gel plates after the development with toluene–acetone–formic acid (3:6:1, v/v) and after the derivatization with DMACA detection reagent. The asterisks (*) in dicate peaks that are only present in the rhizomes of Japanese and Bohemian knotweed.

Figure 7

Comparison of the means of the total peak areas of proanthocyanidins (blue bands in chromatograms) for STSs from rhizomes of Japanese (JK), Bohemian (BK), and giant (GK) knotweed. The mean of the total peak areas was calculated from the total peak areas of the videodensitograms of two equal applications of the same STS on the HPTLC silica gel plate (Figure 3) after the development with toluene–acetone–formic acid (3:6:1, v/v) and after the derivatization with DMACA detection reagent.

Figure 8

The MS spectra obtained by HPTLC—MS analysis of the STS from Bohemian knotweed rhizomes on HPTLC diol F_254S plate pre-developed and developed with acetonitrile. The bolded m/z values in the MS spectra belong to B-type proanthocyanidins and their gallates eluted from the underivatized part of the plate with acetonitrile–methanol (2:1, v/v). A narrow derivatized (DMACA reagent) part of the plate with blue-colored proanthocyanidins zones was used for the proper positioning of the elution head of the TLC—MS interface.

Figure 9

The MS spectra obtained by HPTLC–MS analysis of STS from giant knotweed rhizomes on the HPTLC diol F_254S plate pre-developed and developed with acetonitrile. The bolded m/z values in the MS spectra belong to B-type proan thocyanidins and their gallates eluted from the underivatized part of the plate with acetonitrile–methanol (2:1, v/v). A narrow derivatized (DMACA reagent) part of the plate with blue-colored proanthocyanidins zones was used for the proper positioning of the elution head of the TLC–MS interface.